Tadafumi Shiraishi

Frameshift mutation in the collagen VI gene causes Ullrich's disease

Patients with Ullrichs disease have generalized muscle weakness, multiple contractures of the proximal joints, and hyperextensibility of the distal joints. Recently, we found a deficiency of collagen VI protein in two patients with Ullrichs disease. In this study, we detected a homozygous 26 bp deletion in exon 14 of the collagen VI alpha 2 gene (COL6A2) in one patient. This mutation causes a frameshift and a premature termination codon, and results in a truncated collagen VI alpha 2 chain. Our data suggest that at least some cases of Ullrichs disease result from recessive mutations in COL6A2.

Inhibition of nonsense‐mediated mRNA decay rescues the phenotype in Ullrich's disease

Nonsense‐mediated mRNA decay (NMD) is an mRNA surveillance system that eliminates aberrant mRNAs containing premature translation termination codons (PTCs). We evaluated the role of NMD in of Ullrichs disease. The patient has a frameshift mutation with a PTC in the collagen VI α2 gene causing the loss of collagen VI and functional defects in extracellular matrix (ECM). The pharmacological block of NMD caused upregulation of the mutant collagen VI α2 subunit, resulting in collagen VI assembly and partially functional ECM formation. Our results suggest that NMD inhibitors can be used as a therapeutic tool to rescue some human genetic diseases exacerbated by NMD.

Electron microscopic abnormalities of skeletal muscle in patients with collagen VI deficiency in Ullrich's disease

Abstract. By electron microscopy, we examined the skeletal muscle from two patients with Ullrichs disease. One patient had a deletion in the collagen VI alpha 2 gene. The muscle biopsy specimens showed no collagen VI immunoreaction, while the expression of collagen IV was increased. Collagen VI is a microfibrillar protein in the extracellular matrix with cell adhesive properties, and collagen IV is a principal component of the basal lamina. Electron microscopy revealed unevenness, extension, and folding of the muscle plasma membrane, and showed thickening and overproduction of the basal lamina. The data show that type VI collagen is certainly one of the important extracellular matrix components maintaining the structural integrity of skeletal muscle, and a defect of the collagen VI protein causes abnormalities of the muscle plasma membrane, dystrophic muscle changes, and up-regulation of collagen IV.

Pathological characteristics of skeletal muscle in Ullrich's disease with collagen VI deficiency

Patients with Ullrichs disease have generalized muscle weakness, multiple contractures of the proximal joints and hyperextensibility of the distal joints. Recently, we found a deficiency of collagen VI protein in skeletal muscle from two patients with Ullrichs disease. In this study, we investigated immunohistochemically the expression of extracellular matrix proteins and various proteins, which are markers for regenerating muscle fibers. Although we have detected the reduction of collagen VI in Ullrichs disease with the two kinds of monoclonal antibodies for the different domains of collagen VI, the remaining immunoreactive material was different between them. This might suggest the presence of incomplete collagen VI protein in the muscle fibers. Furthermore, we found that very small muscle fibers in the patients with Ullrichs disease showed marked expression of desmin, neural cell adhesion molecule and neonatal myosin heavy chain, which is a characteristic finding of regenerating fibers, however, they showed poor expression of developmental myosin heavy chain and thrombomodulin. The present findings suggest that abnormal regeneration or maturation processes are involved in the pathogenesis of dystrophic muscle changes at least in the advanced stage of Ullrichs disease.

Inhibition of SMG-8, a subunit of SMG-1 kinase, ameliorates nonsense-mediated mRNA decay-exacerbated mutant phenotypes without cytotoxicity

Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance mechanism that eliminates aberrant mRNAs containing premature termination codons (PTCs). NMD inhibits the production of aberrant proteins that still retain, at least in part, wild-type function as well as dominant-negative peptides. Therefore, the selective inhibition of NMD has the potential to ameliorate NMD-exacerbated mutant phenotypes. However, we do not have sufficient knowledge of how to effectively suppress NMD with minimum cytotoxic effects. In this study, we aimed to identify NMD-related factors that can be targeted to efficiently inhibit NMD without causing significant cytotoxicity to restore the levels of truncated but partially functional proteins. We evaluated the knockdown of 15 NMD components in Ullrich congenital muscular dystrophy fibroblasts, which have a homozygous frameshift mutation causing a PTC in the collagen type VI α 2 gene. Of the 15 NMD factors tested, knockdown of SMG-8 produced the best effect for restoring defective mRNA and protein levels without affecting cell growth, cell-cycle progression, or endoplasmic reticulum stress. The efficacy of SMG-8 knockdown to improve the mutant phenotype was confirmed using another cell line, from a cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy patient who carries a PTC-containing mutation in HtrA serine peptidase 1. Our results suggest that SMG-8 is an appropriate target for inhibiting NMD to improve NMD-exacerbated mutant phenotypes. NMD inhibition by knockdown of SMG-8 may also be useful to induce synergy in combining the use of read-through drugs for patients with nonsense mutation-associated diseases.

Abnormal expression of proteoglycans in Ullrich's disease with collagen VI deficiency.

Patients with Ullrichs disease have generalized muscle weakness, multiple contractures of the proximal joints, and hyperextensibility of the distal joints. Recently we found a marked reduction of fibronectin receptors in the skin and cultured fibroblasts of two patients with Ullrichs disease with collagen VI deficiency, and speculated that an abnormality of cell adhesion may be involved in the pathogenesis of the disease. In this study, we investigated the expression of proteoglycans and adhesion molecules in Ullrichs disease and other muscle diseases. We found a reduction of NG2 proteoglycan in the membrane of skeletal muscle but not in the skin in Ullrichs disease. By contrast, we found the upregulation of tenascin C in the extracellular matrix of skeletal muscle in Ullrichs disease. Our findings suggest that abnormal expression of proteoglycans and adhesion molecules may be involved in the pathogenesis of the dystrophic muscle changes in Ullrichs disease. Muscle Nerve, 2006

Fibronectin receptor reduction in skin and fibroblasts of patients with Ullrich's disease

Ullrichs disease is a congenital muscular dystrophy characterized clinically by generalized muscle weakness, multiple contractures of the proximal joints, and hyperextensibility of the distal joints. Recent studies have demonstrated that collagen VI is deficient in the muscles of patients with Ullrichs disease, and some cases result from recessive mutations of the collagen VIα2 gene (COL6A2). Fibronectin is one of the main components of the extracellular matrix (ECM) and associates with a variety of other matrix molecules including collagen. The behavior of fibronectin on cells is mediated by fibronectin receptors, members of the integrin family. We studied the expression of fibronectin receptors and fibronectin in patients with Ullrichs disease, and found a marked reduction of fibronectin receptors in the ECM of skin and cultured fibroblasts of these patients. These results suggest that collagen VI deficiency may lead to the reduction of fibronectin receptors and that an abnormality of cell adhesion may be involved in the pathogenesis of Ullrichs disease.

Capillary changes in skeletal muscle of patients with Ullrich's disease with collagen VI deficiency

We examined the capillaries in muscle biopsy specimens from two patients with Ullrichs disease with collagen VI deficiency by light and electron microscopy. Collagen VI plays an important role in platelet aggregation for binding von Willebrand factor. Using immunohistochemistry, collagen VI was shown to be absent on capillaries from patients with Ullrichs disease, while von Willebrand factor, collagen IV, and vascular endothelial growth factor were normally expressed. Electron microscopy revealed narrow lumens, large nuclei in endothelial cells, and fenestration of a capillary. The number of pinocytotic vesicles per unit endothelial cytoplasm was increased. The cytoplasm of endothelial cells was strongly stained with uranyl acetate and lead citrate. Replication of the capillary basement membrane was observed. On the other hand, easy bleeding and coagulation were not observed in the two patients. These findings suggested that the collagen VI deficiency might have caused the electron microscopic changes of capillaries, while the function of the capillaries is apparently retained.

Expression of midkine in regenerating skeletal muscle fibers and cultured myoblasts of human skeletal muscle

Midkine is a member of a family of developmentally regulated neurotrophic and heparin-binding growth factors. The expression of midkine was examined immunohistochemically in biopsied muscle specimens from patients with various myopathies. The intense immunoreactivity of midkine was observed in the sarcoplasm at an early stage regenerating fibers of small diameter. Midkine immunoreactivity was also detected in cultured human skeletal muscle myoblasts from the proliferation stage to the differentiation stage. These results suggest that midkine may be an important skeletal muscle growth-promoting factor that participates in the growth process during skeletal muscle development, proliferation, and differentiation, and in the skeletal muscle regeneration in various neuromuscular disorders. This is the first report demonstrating the presence of midkine in skeletal muscle cells.

Different pattern of HSP47 expression in skeletal muscle of patients with neuromuscular diseases.

Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is involved in the processing and secretion of procollagens, and its expression is increased in various fibrotic diseases. However, its involvement in muscle diseases is unknown. In this study, we analyzed HSP47 expression in muscular dystrophies and other muscle diseases. We found an overexpression of HSP47 in fibrous connective tissue and in the adjacent muscle membrane in various muscular dystrophies. However, in Ullrich congenital muscular dystrophy (UCMD), the overexpression of HSP47 was found only in the connective tissue, and not in the muscle membrane. The overexpression of HSP47 was found only in the muscle membrane in the case of active inflammatory myopathy. In particular, HSP47 was strongly expressed in the membrane of regenerating fibers. We found that HSP47 in the muscle membrane locates in the basement membrane with confocal microscopy. Our findings suggest that HSP47 may be involved in the repair or regeneration of muscle fibers in addition to the fibrotic change in the connective tissue.