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Mon-li Chu - One of the best experts on this subject based on the ideXlab platform.
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A Mouse Model for Dominant Collagen VI Disorders HETEROZYGOUS DELETION OF COL6A3 EXON 16
The Journal of biological chemistry, 2014Co-Authors: Te Cheng Pan, David E Birk, Raimund Wagener, Sudheer Kumar Gara, Rui Zhu Zhang, Machiko Arita, Sasha Bogdanovich, Sheila M. Adams, Tejvior S. Khurana, Mon-li ChuAbstract:Dominant and recessive mutations in collagen VI genes, COL6A1, COL6A2, and COL6A3, cause a continuous spectrum of disorders characterized by muscle weakness and connective tissue abnormalities ranging from the severe Ullrich congenital muscular dystrophy to the mild Bethlem myopathy. Herein, we report the development of a mouse model for dominant collagen VI disorders by deleting exon 16 in the COL6A3 gene. The resulting heterozygous mouse, COL6A3+/d16, produced comparable amounts of normal COL6A3 mRNA and a mutant transcript with an in-frame deletion of 54 bp of triple-helical coding sequences, thus mimicking the most common molecular defect found in dominant Ullrich congenital muscular dystrophy patients. Biosynthetic studies of mutant fibroblasts indicated that the mutant α3(VI) collagen protein was produced and exerted a dominant-negative effect on collagen VI microfibrillar assembly. The distribution of the α3(VI)-like chains of collagen VI was not altered in mutant mice during development. The COL6A3+/d16 mice developed histopathologic signs of myopathy and showed ultrastructural alterations of mitochondria and sarcoplasmic reticulum in muscle and abnormal collagen fibrils in tendons. The COL6A3+/d16 mice displayed compromised muscle contractile functions and thereby provide an essential preclinical platform for developing treatment strategies for dominant collagen VI disorders.
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Clinical Significance of Serum COL6A3 in Pancreatic Ductal Adenocarcinoma
Journal of Gastrointestinal Surgery, 2014Co-Authors: Christopher Y. Kang, Mon-li Chu, Jonathan Wang, Dierdre Axell-house, Pranay Soni, Galina Chipitsyna, Konrad Sarosiek, Jocelyn Sendecki, Terry Hyslop, Mazhar Al-zoubiAbstract:Type VI collagen (COL6) forms a microfibrillar network often associated with type I collagen and constitutes a major component of the desmoplastic reaction in pancreatic ductal adenocarcinoma (PDA). We have demonstrated recently that the α 3 chain of COL6, COL6A3, is highly expressed in PDA tissue and undergoes tumor-specific alternative splicing. In this study, we investigated the diagnostic value and clinical significance of circulating COL6A3 protein and mRNA in PDA. COL6A3 levels in sera from patients with PDA ( n = 44), benign lesions ( n = 46) and age-matched healthy volunteers ( n = 30) were analyzed by enzyme-linked immunosorbent assays (ELISA). Predictive abilities of COL6A3 were examined using receiver operating characteristic (ROC) curves from logistic regression models for PDA versus normal or benign serum levels. Expression levels were correlated with clinicopathological parameters. Real-time PCR was used to analyze the presence of COL6A3 mRNA containing alternative spliced exons E3, E4, and E6. Circulating COL6A3 protein levels were significantly elevated in PDA patients when compared to healthy sera ( p = 0.0001) and benign lesions ( p = 0.0035). The overall area under the ROC was 0.975. Log(COL6A3) alone provided good discrimination between PDA and benign lesions (area under the curve (AUC) = 0.817), but combined with CA19-9 provided excellent discrimination (AUC = 0.904). Interestingly, high COL6A3 serum levels were significantly associated with perineural invasion and cigarette smoking. Combined E3, E4, and E6 serum RNA values provided good sensitivity but low specificity. Our data demonstrate for the first time the potential clinical significance of circulating COL6A3 in the diagnosis of pancreatic malignancy.
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COL6A3 protein deficiency in mice leads to muscle and tendon defects similar to human collagen vi congenital muscular dystrophy
Journal of Biological Chemistry, 2013Co-Authors: Te Cheng Pan, David E Birk, Carsten G. Bönnemann, Rui Zhu Zhang, Machiko Arita, Sasha Bogdanovich, Tejvior S. Khurana, Dessislava Markova, Yejia Zhang, Mon-li ChuAbstract:Collagen VI is a ubiquitously expressed extracellular microfibrillar protein. Its most common molecular form is composed of the α1(VI), α2(VI), and α3(VI) collagen α chains encoded by the COL6A1, COL6A2, and COL6A3 genes, respectively. Mutations in any of the three collagen VI genes cause congenital muscular dystrophy types Bethlem and Ullrich as well as intermediate phenotypes characterized by muscle weakness and connective tissue abnormalities. The α3(VI) collagen α chain has much larger N- and C-globular domains than the other two chains. Its most C-terminal domain can be cleaved off after assembly into microfibrils, and the cleavage product has been implicated in tumor angiogenesis and progression. Here we characterize a COL6A3 mutant mouse that expresses a very low level of a non-functional α3(VI) collagen chain. The mutant mice are deficient in extracellular collagen VI microfibrils and exhibit myopathic features, including decreased muscle mass and contractile force. Ultrastructurally abnormal collagen fibrils were observed in tendon, but not cornea, of the mutant mice, indicating a distinct tissue-specific effect of collagen VI on collagen I fibrillogenesis. Overall, the mice lacking normal α3(VI) collagen chains displayed mild musculoskeletal phenotypes similar to mice deficient in the α1(VI) collagen α chain, suggesting that the cleavage product of the α3(VI) collagen does not elicit essential functions in normal growth and development. The COL6A3 mouse mutant lacking functional α3(VI) collagen chains thus serves as an animal model for COL6A3-related muscular dystrophy.
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tumor specific expression and alternative splicing of the COL6A3 gene in pancreatic cancer
Surgery, 2011Co-Authors: Hwyda A Arafat, Te Cheng Pan, Rui Zhu Zhang, Galina Chipitsyna, Melissa Lazar, Khalifa Salem, Qiaoke Gong, Charles J Yeo, Mon-li ChuAbstract:Background Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease; a prominent desmoplastic reaction is a defining characteristic. Fibrillar collagens, such as collagen I and to a lesser extent, collagens III and V, comprise the majority of this stromal fibrosis. Type VI collagen (COL6) forms a microfibrillar network associated with type I collagen fibrils. The expression of COL6 has been linked with inflammation and survival. Importantly, tumor-specific alternative splicing in COL6A3 has been identified in several cancers by genome exon arrays. We evaluated the expression and localization of COL6A3 in PDA and premalignant lesions and explored the presence of alternative splicing events. Methods We analyzed paired PDA–normal (n = 18), intraductal papillary mucinous neoplasms (IPMN; n = 5), pancreatic cystadenoma (n = 5), and 8 PDA cell lines with reverse transcriptase polymerase chain reaction, using unique primers that identify total COL6A3 gene and alternative splicing sites in several of its exons. Western blot analysis and immunohistochemistry were used to analyze the expression levels and localization of COL6A3 protein in the different lesions, and in 2 animal models of PDA. Results COL6A3 protein levels were significantly upregulated in 77% of the paired PDA-adjacent tissue examined. COL6A3 was mainly present in the desmoplastic stroma of PDA, with high deposition around the malignant ducts and in between the sites of stromal fatty infiltration. Analysis of the COL6A3 splice variants showed tumor-specific consistent inclusion of exons 3 and 6 in 17 of the 18 (94%) paired PDA–adjacent tissues. Inclusion of exon 4 was exclusively tumor specific, with barely detectable expression in the adjacent tissues. IPMN and pancreatic cystadenomas showed no expression of any of the examined exons. Total COL6A3 mRNA and exon 6 were identified in 6 PDA cell lines, but only 2 cell lines (MIA PACA-2 and ASPC-1) expressed exons 3 and 4. In both the xenograft and transgenic models of PDA, COL6A3 immunoreactivity was present in the stroma and some PDA cells. Conclusion We have described, for the first time, a dynamic process of tumor-specific alternative splicing in several exons of stromal COL6A3. Alternatively spliced proteins may contribute to the etiology or progression of cancer and may serve as markers for cancer diagnosis. Identification of COL6A3 isoforms as PDA-specific provides the basis for future studies to explore the oncogenic and diagnostic potential of these alternative splicing events.
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recessive col6a2 c globular missense mutations in ullrich congenital muscular dystrophy role of the c2a splice variant
Journal of Biological Chemistry, 2010Co-Authors: Rui Zhu Zhang, Carsten G. Bönnemann, Te Cheng Pan, Suely Kazue Nagahashi Marie, Dessislava Markova, Y Zou, Andrzej Fertala, Stefano Squarzoni, Umbertina Conti Reed, Mon-li ChuAbstract:Ullrich congenital muscular dystrophy (UCMD) is a disabling and life-threatening disorder resulting from either recessive or dominant mutations in genes encoding collagen VI. Although the majority of the recessive UCMD cases have frameshift or nonsense mutations in COL6A1, COL6A2, or COL6A3, recessive structural mutations in the COL6A2 C-globular region are emerging also. However, the underlying molecular mechanisms have remained elusive. Here we identified a homozygous COL6A2 E624K mutation (C1 subdomain) and a homozygous COL6A2 R876S mutation (C2 subdomain) in two UCMD patients. The consequences of the mutations were investigated using fibroblasts from patients and cells stably transfected with the mutant constructs. In contrast to expectations based on the clinical severity of these two patients, secretion and assembly of collagen VI were moderately affected by the E624K mutation but severely impaired by the R876S substitution. The E624K substitution altered the electrostatic potential of the region surrounding the metal ion-dependent adhesion site, resulting in a collagen VI network containing thick fibrils and spots with densely packed microfibrils. The R876S mutation prevented the chain from assembling into triple-helical collagen VI molecules. The minute amount of collagen VI secreted by the R876S fibroblasts was solely composed of a faster migrating chain corresponding to the C2a splice variant with an alternative C2 subdomain. In transfected cells, the C2a splice variant was able to assemble into short microfibrils. Together, the results suggest that the C2a splice variant may functionally compensate for the loss of the normal COL6A2 chain when mutations occur in the C2 subdomain.
Carsten G. Bönnemann - One of the best experts on this subject based on the ideXlab platform.
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exon skipping oligonucleotides restore functional collagen vi by correcting a common col6a1 mutation in ullrich cmd
Molecular therapy. Nucleic acids, 2020Co-Authors: Sara Aguti, Carsten G. Bönnemann, Francesco Muntoni, Pierpaolo Ala, Veronique Bolduc, Mark Turmaine, Haiyan ZhouAbstract:Collagen VI-related congenital muscular dystrophies (COL6-CMDs) are the second most common form of congenital muscular dystrophy. Currently, there is no effective treatment available. COL6-CMDs are caused by recessive or dominant mutations in one of the three genes encoding for the α chains of collagen type VI (COL6A1, COL6A2, and COL6A3). One of the most common mutations in COL6-CMD patients is a de novo deep intronic c.930+189C > T mutation in COL6A1 gene. This mutation creates a cryptic donor splice site and induces incorporation of a novel in-frame pseudo-exon in the mature transcripts. In this study, we systematically evaluated the splice switching approach using antisense oligonucleotides (ASOs) to correct this mutation. Fifteen ASOs were designed using the RNA-tiling approach to target the misspliced pseudo-exon and its flanking sequences. The efficiency of ASOs was evaluated at RNA, protein, and structural levels in skin fibroblasts established from four patients carrying the c.930+189C > T mutation. We identified two additional lead ASO candidates that efficiently induce pseudo-exon exclusion from the mature transcripts, thus allowing for the restoration of a functional collagen VI microfibrillar matrix. Our findings provide further evidence for ASO exon skipping as a therapeutic approach for COL6-CMD patients carrying this common intronic mutation.
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lower extremity muscle involvement in the intermediate and bethlem myopathy forms of col6 related dystrophy and duchenne muscular dystrophy a cross sectional study
Journal of neuromuscular diseases, 2020Co-Authors: Abhinandan Batra, Carsten G. Bönnemann, Reghan A Foley, Jahannaz Dastgir, Donovan J Lott, Rebecca J Willcocks, Sean C Forbes, William T Triplett, Pomi Yun, Krista VandenborneAbstract:Collagen VI-related dystrophies (COL6-RDs) and Duchenne muscular dystrophy (DMD) cause progressive muscle weakness and disability. COL6-RDs are caused by mutations in the COL6 genes (COL6A1, COL6A2 and COL6A3) encoding the extracellular matrix protein collagen VI, and DMD is caused by mutations in the DMD gene encoding the cytoplasmic protein dystrophin. Both COL6-RDs and DMD are characterized by infiltration of the muscles by fatty and fibrotic tissue. This study examined the effect of disease pathology on skeletal muscles in lower extremity muscles of COL6-RDs using timed functional tests, strength measures and qualitative/ quantitative magnetic resonance imaging/spectroscopy measures (MRI/MRS) in comparison to unaffected (control) individuals. Patients with COL6-RD were also compared to age and gender matched patients with DMD.Patients with COL6-RD presented with a typical pattern of fatty infiltration of the muscle giving rise to an apparent halo effect around the muscle, while patients with DMD had evidence of fatty infiltration throughout the muscle areas imaged. Quantitatively, fat fraction, and transverse relaxation time (T2) were elevated in both COL6-RD and DMD patients compared to unaffected (control) individuals. Patients with COL6-RD had widespread muscle atrophy, likely contributing to weakness. In contrast, patients with DMD revealed force deficits even in muscle groups with increased contractile areas.
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O.1 Collagen XII as a new disease gene for Bethlem-like myopathy
Neuromuscular Disorders, 2013Co-Authors: Debbie Hicks, Carsten G. Bönnemann, James J Collins, Yaqun Zou, E Martoni, G. Torabi Farsani, Steve Laval, A. Shah, Manuel Koch, Hanns LochmüllerAbstract:Bethlem myopathy (BM) is a slowly progressive disorder characterized by contractures and a proximal myopathy, which can be caused by mutations in one of the collagen VI genes (COL6A1, COL6A2, and COL6A3), although genetic heterogeneity is predicted to be as high as 50%. Using whole exome sequencing (WES) we have identified mutations in a novel gene, COL12A1, a member of the FACIT collagens (fibril-associated collagens with interrupted triple helices) in five individuals from two families. In eighteen additional families we have excluded 12 other genes which were considered candidates for causative genes in non-collagen VI BM based on their function, including known binding partners of, and enzymes involved in correct post translational modification, assembly and secretion of collagen VI. Both families carry dominant mutations with a clinical phenotype indistinguishable from classical BM, apart from an intriguing improvement in disease course over time in one of the families and a different pattern of selective muscle involvement on MRI. Family 1 has a single-base substitution that leads to the replacement of one glycine residue in the triple helical domain, breaking the Gly-X-Y repeating pattern, which is known to destabilize the triple helix through a local disruption in hydrogen bonding. Family 2 has a missense mutation which creates a mutant protein with an unpaired cysteine residue. Deficiency at the protein level is confirmed in both families by the intracellular retention of collagen XII in patient dermal fibroblasts. Upregulation of genes associated with the endoplasmic reticulum-associated protein degradation (ERAD) pathway and swollen, dysmorphic rough-ER as determined by electron microscopy in three members of Family 2 lead to the hypothesis that the unfolded protein response (UPR) contributes to the disease pathomechanism in this family. We suggest that the spectrum of causative genes in BM be extended from the COL6A1-A3 genes to include COL12A1.
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COL6A3 protein deficiency in mice leads to muscle and tendon defects similar to human collagen vi congenital muscular dystrophy
Journal of Biological Chemistry, 2013Co-Authors: Te Cheng Pan, David E Birk, Carsten G. Bönnemann, Rui Zhu Zhang, Machiko Arita, Sasha Bogdanovich, Tejvior S. Khurana, Dessislava Markova, Yejia Zhang, Mon-li ChuAbstract:Collagen VI is a ubiquitously expressed extracellular microfibrillar protein. Its most common molecular form is composed of the α1(VI), α2(VI), and α3(VI) collagen α chains encoded by the COL6A1, COL6A2, and COL6A3 genes, respectively. Mutations in any of the three collagen VI genes cause congenital muscular dystrophy types Bethlem and Ullrich as well as intermediate phenotypes characterized by muscle weakness and connective tissue abnormalities. The α3(VI) collagen α chain has much larger N- and C-globular domains than the other two chains. Its most C-terminal domain can be cleaved off after assembly into microfibrils, and the cleavage product has been implicated in tumor angiogenesis and progression. Here we characterize a COL6A3 mutant mouse that expresses a very low level of a non-functional α3(VI) collagen chain. The mutant mice are deficient in extracellular collagen VI microfibrils and exhibit myopathic features, including decreased muscle mass and contractile force. Ultrastructurally abnormal collagen fibrils were observed in tendon, but not cornea, of the mutant mice, indicating a distinct tissue-specific effect of collagen VI on collagen I fibrillogenesis. Overall, the mice lacking normal α3(VI) collagen chains displayed mild musculoskeletal phenotypes similar to mice deficient in the α1(VI) collagen α chain, suggesting that the cleavage product of the α3(VI) collagen does not elicit essential functions in normal growth and development. The COL6A3 mouse mutant lacking functional α3(VI) collagen chains thus serves as an animal model for COL6A3-related muscular dystrophy.
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the collagen vi related myopathies ullrich congenital muscular dystrophy and bethlem myopathy
Handbook of Clinical Neurology, 2011Co-Authors: Carsten G. BönnemannAbstract:Abstract Mutations in the genes COL6A1, COL6A2, and COL6A3, coding for three α chains of collagen type VI, underlie a spectrum of myopathies, ranging from the severe congenital muscular dystrophy-type Ullrich (UCMD) to the milder Bethlem myopathy (BM), with disease manifestations of intermediate severity in between. UCMD is characterized by early‐onset weakness, associated with pronounced distal joint hyperlaxity and the early onset or early progression of more proximal contractures. In the most severe cases ambulation is not achieved, or it may be achieved only for a limited period of time. BM may be of early or later onset, but is milder in its manifestations, typically allowing for ambulation well into adulthood, whereas typical joint contractures are frequently prominent. A genetic spectrum is emerging, with BM being caused mostly by dominantly acting mutations, although rarely recessive inheritance of BM is also possible, whereas both dominantly as well as recessively acting mutations underlie UCMD.
Te Cheng Pan - One of the best experts on this subject based on the ideXlab platform.
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A Mouse Model for Dominant Collagen VI Disorders HETEROZYGOUS DELETION OF COL6A3 EXON 16
The Journal of biological chemistry, 2014Co-Authors: Te Cheng Pan, David E Birk, Raimund Wagener, Sudheer Kumar Gara, Rui Zhu Zhang, Machiko Arita, Sasha Bogdanovich, Sheila M. Adams, Tejvior S. Khurana, Mon-li ChuAbstract:Dominant and recessive mutations in collagen VI genes, COL6A1, COL6A2, and COL6A3, cause a continuous spectrum of disorders characterized by muscle weakness and connective tissue abnormalities ranging from the severe Ullrich congenital muscular dystrophy to the mild Bethlem myopathy. Herein, we report the development of a mouse model for dominant collagen VI disorders by deleting exon 16 in the COL6A3 gene. The resulting heterozygous mouse, COL6A3+/d16, produced comparable amounts of normal COL6A3 mRNA and a mutant transcript with an in-frame deletion of 54 bp of triple-helical coding sequences, thus mimicking the most common molecular defect found in dominant Ullrich congenital muscular dystrophy patients. Biosynthetic studies of mutant fibroblasts indicated that the mutant α3(VI) collagen protein was produced and exerted a dominant-negative effect on collagen VI microfibrillar assembly. The distribution of the α3(VI)-like chains of collagen VI was not altered in mutant mice during development. The COL6A3+/d16 mice developed histopathologic signs of myopathy and showed ultrastructural alterations of mitochondria and sarcoplasmic reticulum in muscle and abnormal collagen fibrils in tendons. The COL6A3+/d16 mice displayed compromised muscle contractile functions and thereby provide an essential preclinical platform for developing treatment strategies for dominant collagen VI disorders.
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COL6A3 protein deficiency in mice leads to muscle and tendon defects similar to human collagen vi congenital muscular dystrophy
Journal of Biological Chemistry, 2013Co-Authors: Te Cheng Pan, David E Birk, Carsten G. Bönnemann, Rui Zhu Zhang, Machiko Arita, Sasha Bogdanovich, Tejvior S. Khurana, Dessislava Markova, Yejia Zhang, Mon-li ChuAbstract:Collagen VI is a ubiquitously expressed extracellular microfibrillar protein. Its most common molecular form is composed of the α1(VI), α2(VI), and α3(VI) collagen α chains encoded by the COL6A1, COL6A2, and COL6A3 genes, respectively. Mutations in any of the three collagen VI genes cause congenital muscular dystrophy types Bethlem and Ullrich as well as intermediate phenotypes characterized by muscle weakness and connective tissue abnormalities. The α3(VI) collagen α chain has much larger N- and C-globular domains than the other two chains. Its most C-terminal domain can be cleaved off after assembly into microfibrils, and the cleavage product has been implicated in tumor angiogenesis and progression. Here we characterize a COL6A3 mutant mouse that expresses a very low level of a non-functional α3(VI) collagen chain. The mutant mice are deficient in extracellular collagen VI microfibrils and exhibit myopathic features, including decreased muscle mass and contractile force. Ultrastructurally abnormal collagen fibrils were observed in tendon, but not cornea, of the mutant mice, indicating a distinct tissue-specific effect of collagen VI on collagen I fibrillogenesis. Overall, the mice lacking normal α3(VI) collagen chains displayed mild musculoskeletal phenotypes similar to mice deficient in the α1(VI) collagen α chain, suggesting that the cleavage product of the α3(VI) collagen does not elicit essential functions in normal growth and development. The COL6A3 mouse mutant lacking functional α3(VI) collagen chains thus serves as an animal model for COL6A3-related muscular dystrophy.
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tumor specific expression and alternative splicing of the COL6A3 gene in pancreatic cancer
Surgery, 2011Co-Authors: Hwyda A Arafat, Te Cheng Pan, Rui Zhu Zhang, Galina Chipitsyna, Melissa Lazar, Khalifa Salem, Qiaoke Gong, Charles J Yeo, Mon-li ChuAbstract:Background Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease; a prominent desmoplastic reaction is a defining characteristic. Fibrillar collagens, such as collagen I and to a lesser extent, collagens III and V, comprise the majority of this stromal fibrosis. Type VI collagen (COL6) forms a microfibrillar network associated with type I collagen fibrils. The expression of COL6 has been linked with inflammation and survival. Importantly, tumor-specific alternative splicing in COL6A3 has been identified in several cancers by genome exon arrays. We evaluated the expression and localization of COL6A3 in PDA and premalignant lesions and explored the presence of alternative splicing events. Methods We analyzed paired PDA–normal (n = 18), intraductal papillary mucinous neoplasms (IPMN; n = 5), pancreatic cystadenoma (n = 5), and 8 PDA cell lines with reverse transcriptase polymerase chain reaction, using unique primers that identify total COL6A3 gene and alternative splicing sites in several of its exons. Western blot analysis and immunohistochemistry were used to analyze the expression levels and localization of COL6A3 protein in the different lesions, and in 2 animal models of PDA. Results COL6A3 protein levels were significantly upregulated in 77% of the paired PDA-adjacent tissue examined. COL6A3 was mainly present in the desmoplastic stroma of PDA, with high deposition around the malignant ducts and in between the sites of stromal fatty infiltration. Analysis of the COL6A3 splice variants showed tumor-specific consistent inclusion of exons 3 and 6 in 17 of the 18 (94%) paired PDA–adjacent tissues. Inclusion of exon 4 was exclusively tumor specific, with barely detectable expression in the adjacent tissues. IPMN and pancreatic cystadenomas showed no expression of any of the examined exons. Total COL6A3 mRNA and exon 6 were identified in 6 PDA cell lines, but only 2 cell lines (MIA PACA-2 and ASPC-1) expressed exons 3 and 4. In both the xenograft and transgenic models of PDA, COL6A3 immunoreactivity was present in the stroma and some PDA cells. Conclusion We have described, for the first time, a dynamic process of tumor-specific alternative splicing in several exons of stromal COL6A3. Alternatively spliced proteins may contribute to the etiology or progression of cancer and may serve as markers for cancer diagnosis. Identification of COL6A3 isoforms as PDA-specific provides the basis for future studies to explore the oncogenic and diagnostic potential of these alternative splicing events.
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recessive col6a2 c globular missense mutations in ullrich congenital muscular dystrophy role of the c2a splice variant
Journal of Biological Chemistry, 2010Co-Authors: Rui Zhu Zhang, Carsten G. Bönnemann, Te Cheng Pan, Suely Kazue Nagahashi Marie, Dessislava Markova, Y Zou, Andrzej Fertala, Stefano Squarzoni, Umbertina Conti Reed, Mon-li ChuAbstract:Ullrich congenital muscular dystrophy (UCMD) is a disabling and life-threatening disorder resulting from either recessive or dominant mutations in genes encoding collagen VI. Although the majority of the recessive UCMD cases have frameshift or nonsense mutations in COL6A1, COL6A2, or COL6A3, recessive structural mutations in the COL6A2 C-globular region are emerging also. However, the underlying molecular mechanisms have remained elusive. Here we identified a homozygous COL6A2 E624K mutation (C1 subdomain) and a homozygous COL6A2 R876S mutation (C2 subdomain) in two UCMD patients. The consequences of the mutations were investigated using fibroblasts from patients and cells stably transfected with the mutant constructs. In contrast to expectations based on the clinical severity of these two patients, secretion and assembly of collagen VI were moderately affected by the E624K mutation but severely impaired by the R876S substitution. The E624K substitution altered the electrostatic potential of the region surrounding the metal ion-dependent adhesion site, resulting in a collagen VI network containing thick fibrils and spots with densely packed microfibrils. The R876S mutation prevented the chain from assembling into triple-helical collagen VI molecules. The minute amount of collagen VI secreted by the R876S fibroblasts was solely composed of a faster migrating chain corresponding to the C2a splice variant with an alternative C2 subdomain. In transfected cells, the C2a splice variant was able to assemble into short microfibrils. Together, the results suggest that the C2a splice variant may functionally compensate for the loss of the normal COL6A2 chain when mutations occur in the C2 subdomain.
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new molecular mechanism for ullrich congenital muscular dystrophy a heterozygous in frame deletion in the col6a1 gene causes a severe phenotype
American Journal of Human Genetics, 2003Co-Authors: Te Cheng Pan, Carsten G. Bönnemann, Rui Zhu Zhang, Suely Kazue Nagahashi Marie, Dominick Sudano, Mon-li ChuAbstract: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.
Rui Zhu Zhang - One of the best experts on this subject based on the ideXlab platform.
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A Mouse Model for Dominant Collagen VI Disorders HETEROZYGOUS DELETION OF COL6A3 EXON 16
The Journal of biological chemistry, 2014Co-Authors: Te Cheng Pan, David E Birk, Raimund Wagener, Sudheer Kumar Gara, Rui Zhu Zhang, Machiko Arita, Sasha Bogdanovich, Sheila M. Adams, Tejvior S. Khurana, Mon-li ChuAbstract:Dominant and recessive mutations in collagen VI genes, COL6A1, COL6A2, and COL6A3, cause a continuous spectrum of disorders characterized by muscle weakness and connective tissue abnormalities ranging from the severe Ullrich congenital muscular dystrophy to the mild Bethlem myopathy. Herein, we report the development of a mouse model for dominant collagen VI disorders by deleting exon 16 in the COL6A3 gene. The resulting heterozygous mouse, COL6A3+/d16, produced comparable amounts of normal COL6A3 mRNA and a mutant transcript with an in-frame deletion of 54 bp of triple-helical coding sequences, thus mimicking the most common molecular defect found in dominant Ullrich congenital muscular dystrophy patients. Biosynthetic studies of mutant fibroblasts indicated that the mutant α3(VI) collagen protein was produced and exerted a dominant-negative effect on collagen VI microfibrillar assembly. The distribution of the α3(VI)-like chains of collagen VI was not altered in mutant mice during development. The COL6A3+/d16 mice developed histopathologic signs of myopathy and showed ultrastructural alterations of mitochondria and sarcoplasmic reticulum in muscle and abnormal collagen fibrils in tendons. The COL6A3+/d16 mice displayed compromised muscle contractile functions and thereby provide an essential preclinical platform for developing treatment strategies for dominant collagen VI disorders.
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COL6A3 protein deficiency in mice leads to muscle and tendon defects similar to human collagen vi congenital muscular dystrophy
Journal of Biological Chemistry, 2013Co-Authors: Te Cheng Pan, David E Birk, Carsten G. Bönnemann, Rui Zhu Zhang, Machiko Arita, Sasha Bogdanovich, Tejvior S. Khurana, Dessislava Markova, Yejia Zhang, Mon-li ChuAbstract:Collagen VI is a ubiquitously expressed extracellular microfibrillar protein. Its most common molecular form is composed of the α1(VI), α2(VI), and α3(VI) collagen α chains encoded by the COL6A1, COL6A2, and COL6A3 genes, respectively. Mutations in any of the three collagen VI genes cause congenital muscular dystrophy types Bethlem and Ullrich as well as intermediate phenotypes characterized by muscle weakness and connective tissue abnormalities. The α3(VI) collagen α chain has much larger N- and C-globular domains than the other two chains. Its most C-terminal domain can be cleaved off after assembly into microfibrils, and the cleavage product has been implicated in tumor angiogenesis and progression. Here we characterize a COL6A3 mutant mouse that expresses a very low level of a non-functional α3(VI) collagen chain. The mutant mice are deficient in extracellular collagen VI microfibrils and exhibit myopathic features, including decreased muscle mass and contractile force. Ultrastructurally abnormal collagen fibrils were observed in tendon, but not cornea, of the mutant mice, indicating a distinct tissue-specific effect of collagen VI on collagen I fibrillogenesis. Overall, the mice lacking normal α3(VI) collagen chains displayed mild musculoskeletal phenotypes similar to mice deficient in the α1(VI) collagen α chain, suggesting that the cleavage product of the α3(VI) collagen does not elicit essential functions in normal growth and development. The COL6A3 mouse mutant lacking functional α3(VI) collagen chains thus serves as an animal model for COL6A3-related muscular dystrophy.
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tumor specific expression and alternative splicing of the COL6A3 gene in pancreatic cancer
Surgery, 2011Co-Authors: Hwyda A Arafat, Te Cheng Pan, Rui Zhu Zhang, Galina Chipitsyna, Melissa Lazar, Khalifa Salem, Qiaoke Gong, Charles J Yeo, Mon-li ChuAbstract:Background Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease; a prominent desmoplastic reaction is a defining characteristic. Fibrillar collagens, such as collagen I and to a lesser extent, collagens III and V, comprise the majority of this stromal fibrosis. Type VI collagen (COL6) forms a microfibrillar network associated with type I collagen fibrils. The expression of COL6 has been linked with inflammation and survival. Importantly, tumor-specific alternative splicing in COL6A3 has been identified in several cancers by genome exon arrays. We evaluated the expression and localization of COL6A3 in PDA and premalignant lesions and explored the presence of alternative splicing events. Methods We analyzed paired PDA–normal (n = 18), intraductal papillary mucinous neoplasms (IPMN; n = 5), pancreatic cystadenoma (n = 5), and 8 PDA cell lines with reverse transcriptase polymerase chain reaction, using unique primers that identify total COL6A3 gene and alternative splicing sites in several of its exons. Western blot analysis and immunohistochemistry were used to analyze the expression levels and localization of COL6A3 protein in the different lesions, and in 2 animal models of PDA. Results COL6A3 protein levels were significantly upregulated in 77% of the paired PDA-adjacent tissue examined. COL6A3 was mainly present in the desmoplastic stroma of PDA, with high deposition around the malignant ducts and in between the sites of stromal fatty infiltration. Analysis of the COL6A3 splice variants showed tumor-specific consistent inclusion of exons 3 and 6 in 17 of the 18 (94%) paired PDA–adjacent tissues. Inclusion of exon 4 was exclusively tumor specific, with barely detectable expression in the adjacent tissues. IPMN and pancreatic cystadenomas showed no expression of any of the examined exons. Total COL6A3 mRNA and exon 6 were identified in 6 PDA cell lines, but only 2 cell lines (MIA PACA-2 and ASPC-1) expressed exons 3 and 4. In both the xenograft and transgenic models of PDA, COL6A3 immunoreactivity was present in the stroma and some PDA cells. Conclusion We have described, for the first time, a dynamic process of tumor-specific alternative splicing in several exons of stromal COL6A3. Alternatively spliced proteins may contribute to the etiology or progression of cancer and may serve as markers for cancer diagnosis. Identification of COL6A3 isoforms as PDA-specific provides the basis for future studies to explore the oncogenic and diagnostic potential of these alternative splicing events.
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recessive col6a2 c globular missense mutations in ullrich congenital muscular dystrophy role of the c2a splice variant
Journal of Biological Chemistry, 2010Co-Authors: Rui Zhu Zhang, Carsten G. Bönnemann, Te Cheng Pan, Suely Kazue Nagahashi Marie, Dessislava Markova, Y Zou, Andrzej Fertala, Stefano Squarzoni, Umbertina Conti Reed, Mon-li ChuAbstract:Ullrich congenital muscular dystrophy (UCMD) is a disabling and life-threatening disorder resulting from either recessive or dominant mutations in genes encoding collagen VI. Although the majority of the recessive UCMD cases have frameshift or nonsense mutations in COL6A1, COL6A2, or COL6A3, recessive structural mutations in the COL6A2 C-globular region are emerging also. However, the underlying molecular mechanisms have remained elusive. Here we identified a homozygous COL6A2 E624K mutation (C1 subdomain) and a homozygous COL6A2 R876S mutation (C2 subdomain) in two UCMD patients. The consequences of the mutations were investigated using fibroblasts from patients and cells stably transfected with the mutant constructs. In contrast to expectations based on the clinical severity of these two patients, secretion and assembly of collagen VI were moderately affected by the E624K mutation but severely impaired by the R876S substitution. The E624K substitution altered the electrostatic potential of the region surrounding the metal ion-dependent adhesion site, resulting in a collagen VI network containing thick fibrils and spots with densely packed microfibrils. The R876S mutation prevented the chain from assembling into triple-helical collagen VI molecules. The minute amount of collagen VI secreted by the R876S fibroblasts was solely composed of a faster migrating chain corresponding to the C2a splice variant with an alternative C2 subdomain. In transfected cells, the C2a splice variant was able to assemble into short microfibrils. Together, the results suggest that the C2a splice variant may functionally compensate for the loss of the normal COL6A2 chain when mutations occur in the C2 subdomain.
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Muscle Interstitial Fibroblasts Are the Main Source of Collagen VI Synthesis in Skeletal Muscle: Implications for Congenital Muscular Dystrophy Types Ullrich and Bethlem
Journal of neuropathology and experimental neurology, 2008Co-Authors: Yaqun Zou, Patrizia Sabatelli, Rui Zhu Zhang, Mon-li Chu, Carsten G. BönnemannAbstract:Mutations in the extracellular matrix molecule collagen VI underlie the congenital muscular dystrophy types Ullrich and Bethlem. Establishing the origin of collagen VI in muscle is important for understanding the pathophysiology of these diseases and for developing future treatment approaches involving cell-specific delivery. Because the cells that produce collagen VI cannot be identified by histologic analysis, we examined the production of collagen VI in pure cultures of primary myogenic cells and muscle interstitial fibroblasts from limb muscle of neonatal mice. Immunofluorescence staining and Western blot analysis revealed secretion and matrix deposition of collagen VI by interstitial fibroblasts but not by myogenic cells in vitro. Using Northern blot and real-time reverse-transcriptase-polymerase chain reaction analysis for the collagen VI genes col6a1 , col6a2 , COL6A3 , transcript levels for the 3 mRNAs were high in interstitial fibroblasts, whereas in primary myogenic cells, they were indistinguishable from background. Furthermore, retention of mutant collagen VI in muscle from 3 patients with collagen VI mutation was identified in interstitial fibroblastic cells but not in their myofibers. These results suggest that interstitial fibroblasts but not myogenic cells contribute significantly to the deposition of collagen VI in the extracellular matrix in skeletal muscle and imply major roles of this cell type and the extracellular matrix in the pathogenesis of these diseases.
Marcy C Speer - One of the best experts on this subject based on the ideXlab platform.
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missense mutation in a von willebrand factor type a domain of the α3 vi collagen gene COL6A3 in a family with bethlem myopathy
Human Molecular Genetics, 1998Co-Authors: Te Cheng Pan, Rui Zhu Zhang, Mon-li Chu, Jeffrey M. Stajich, Rup Tandan, Timothy J. Fries, Jeffery M. Vance, Kristi D. Viles, Margaret A Pericakvance, Marcy C SpeerAbstract:The Bethlem myopathy is a rare autosomal dominant proximal myopathy characterized by early childhood onset and joint contractures. Evidence for linkage and genetic heterogeneity has been established, with the majority of families linked to 21q22.3 and one large family linked to 2q37, implicating the three type VI collagen subunit genes, COL6A1 (chromosome 21), COL6A2 (chromosome 21) and COL6A3 (chromosome 2) as candidate genes. Mutations of the invariant glycine residues in the triple-helical domain-coding region of COL6A1 and COL6A2 have been reported previously in the chromosome 21-linked families. We report here the identification of a G∀ A mutation in the N-terminal globular domain-coding region of COL6A3 in a large American pedigree (19 affected, 12 unaffected), leading to the substitution of glycine by glutamic acid in the N2 motif, which is homologous to the type A domains of the von Willebrand factor. This mutation segregated to all affected family members, to no unaffected family members, and was not identified in 338 unrelated Caucasian control chromosomes. Thus mutations in either the triple-helical domain or the globular domain of type VI collagen appear to cause Bethlem myopathy.
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type vi collagen mutations in bethlem myopathy an autosomal dominant myopathy with contractures
Nature Genetics, 1996Co-Authors: G J Jobsis, H Keizers, Jeroen Vreijling, M De Visser, Marcy C Speer, Ruud A Wolterman, Frank Baas, P A BolhuisAbstract:Among the diverse family of collagens, the widely expressed microfibrillar type VI collagen is believed to play a role in bridging cells with the extracellular matrix. Several observations imply substrate properties for cell attachment1 as well as association with major collagen fibers2. Previously, we have established genetic linkage between the genes encoding the three constituent α-chains of type VI collagen and Bethlem myopathy3–5. A distinctive feature of this autosomal dominant disorder consists of contractures of multiple joints in addition to generalized muscular weakness and wasting6–10. Nine kindreds show genetic linkage to the COL6A1–COL6A2 cluster on chromosome 21q22.3 (refs 3,4; manuscript submitted) whereas one family shows linkage to markers on chromosome 2q37 close to COL6A3 (ref. 5). Sequence analysis in four families reveals a mutation in COL6A1 in one and a COL6A2 mutation in two other kindreds. Both mutations disrupt the Gly-X-Y motif of the triple helical domain by substitution of Gly for either Val or Ser. Analogous to the putative perturbation of the anchoring function of the dystrophin-associated complex in congenital muscular dystrophy with mutations in the α2-subunit of laminin, our observations suggest a similar mechanism in Bethlem myopathy.
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Evidence for Locus Heterogeneity in the Bethlem Myopathy and Linkage to 2q37
Human molecular genetics, 1996Co-Authors: Marcy C Speer, G J Jobsis, Jeffrey M. Stajich, Rup Tandan, Nagesh Rao, Timothy J. Fries, Pieter A. Bolhuis, Jeffery M. Vance, Kristi D. Viles, Karen SheffieldAbstract:The Bethlem myopathy, a childhood onset autosomal dominant myopathy with joint contractures, has recently been localized to 21q in a series of Dutch families and the alpha 1 and alpha 2 subunits of type VI collagen (COL6A1 and COL6A2) have been postulated as candidate genes. We investigate a large family of French Canadian descent (family 1489) in which the Bethlem myopathy is segregating. Family 1489 is unlinked to the region of interest on 21q, thus demonstrating locus heterogeneity within the Bethlem myopathy classification. In view of the localization of the genes coding the alpha 1 and alpha 2 subunits of type VI collagen on chromosome 21q, we carried out linkage analysis on chromosome 2q where the alpha 3 subunit of type VI collagen has been localized. We demonstrate linkage to markers in this region, define the region of disease gene localization, and confirm by FISH analysis that COL6A3 is located within the interval of interest making COL6A3 a feasible candidate gene for the Bethlem myopathy.