Altered TDP-43-dependent splicing in HSPB8-related distal hereditary motor neuropathy and myofibrillar myopathy.

BACKGROUND AND PURPOSE: Mutations in the small heat-shock protein 22 gene (HSPB8) have been associated with Charcot-Marie-Tooth disease type 2L, distal hereditary motor neuropathy (dHMN) type IIa and, more recently, distal myopathy/myofibrillar myopathy (MFM) with protein aggregates and TDP-43 inclusions. The aim was to report a novel family with HSPB8K141E -related dHMN/MFM and to investigate, in a patient muscle biopsy, whether the presence of protein aggregates was paralleled by altered TDP-43 function. METHODS: We reviewed clinical and genetic data. We assessed TDP-43 expression by qPCR and alternative splicing of four previously validated direct TDP-43 target exons in four genes by reverse transcriptase-polymerase chain reaction. RESULTS: The triplets and their mother presented in the second to third decade of life with progressive weakness affecting distal and proximal lower limb and truncal muscles. Nerve conduction study showed a motor axonal neuropathy. The clinical features, moderately raised creatin kinase levels, selective pattern of muscle involvement on magnetic resonance imaging and pathological changes on muscle biopsy, including the presence of protein aggregates, supported the diagnosis of a contemporary primary muscle involvement. In affected muscle tissue we observed a consistent alteration of TDP-43-dependent splicing in three out of four TDP-43-target transcripts (POLDIP3, FNIP1 and BRD8), as well as a significant decrease of TDP-43 mRNA levels. CONCLUSIONS: Our study confirmed the role of mutated HSPB8 as a cause of a combined neuromuscular disorder encompassing dHMN and MFM with protein aggregates. We identified impaired RNA metabolism, secondary to TDP-43 loss of function, as a possible pathological mechanism of HSPB8K141E toxicity, leading to muscle and nerve degeneration.

Fatal hepatic hemorrhage by peliosis hepatis in X-linked myotubular myopathy: a case report.

We report a 5-year-old boy with X-linked myotubular myopathy complicated by peliosis hepatis. At birth, he was affected with marked generalized muscle hypotonia and weakness, which required permanent ventilatory support, and was bedridden for life. He died of acute fatal hepatic hemorrhage after using a mechanical in-exsufflator. Peliosis hepatis, defined as multiple, variable-sized, cystic blood-filled spaces through the liver parenchyma, was confirmed by autopsy. To avoid fatal hepatic hemorrhage by peliosis hepatis, routine hepatic function tests and abdominal imaging tests should be performed for patients with X-linked myotubular myopathy, especially at the time of using artificial respiration.

Characterization of the Asian myopathy patients with VCP mutations.

BACKGROUND AND PURPOSE: Mutations in the valosin-containing protein (VCP) gene are known to cause inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD) and familial amyotrophic lateral sclerosis (ALS). Despite an increasing number of clinical reports, only one Asian family with IBMPFD has been described. METHODS: To characterize patients with VCP mutations, we screened a total of 152 unrelated Asian families who were suspected to have rimmed vacuolar myopathy. RESULTS: We identified VCP mutations in seven patients from six unrelated Asian families. Five different missense mutations were found, including a novel p.Ala439Pro substitution. All patients had adult-onset progressive muscle wasting with variable involvement of axial, proximal, and distal muscles. Two of seven patients were suggested to have mild brain involvement including cerebellar ataxia, and only one showed radiological findings indicating a change in bone. Findings from skeletal muscle indicated mixed neurogenic and myogenic changes, fibers with rimmed vacuoles, and the presence of cytoplasmic and nuclear inclusions. These inclusions were immunopositive for VCP, ubiquitin, transactivation response DNA-binding protein 43, and also histone deacetylase 6 (HDAC6), of which function is regulated by VCP. Evidence of early nuclear and mitochondrial damage was also characteristic. CONCLUSIONS: Valosin-containing protein mutations are not rare in Asian patients, and gene analysis should be considered for patients with adult-onset rimmed vacuolar myopathy with neurogenic changes. A wide variety of central and peripheral nervous system symptoms coupled with rare bone abnormalities may complicate diagnosis.

Congenital neuromuscular disease with uniform type 1 fiber and RYR1 mutation.

BACKGROUND: Congenital neuromuscular disease with uniform type 1 fiber (CNMDU1) is a rare form of congenital myopathy, which is pathologically diagnosed by the presence of more than 99% of type 1 fiber, with no specific structural changes. Its pathogenic mechanism is still unknown. We recently reported that almost all patients with central core disease (CCD) with ryanodine receptor 1 gene (RYR1) mutations in the C-terminal domain had type 1 fibers, nearly exclusively, in addition to typical central cores. OBJECTIVE: To investigate whether CNMDU1 is associated with RYR1 mutation. METHODS: We studied 10 unrelated Japanese patients who were diagnosed to have CNMDU1 based on clinical features and muscle pathology showing more than 99% type 1 muscle fibers. We extracted genomic DNA from frozen muscles and directly sequenced all 106 exons and their flanking intron-exon boundaries of RYR1. RESULTS: Four of 10 patients had a heterozygous mutation, three missense and one deletion, all in the C-terminal domain of RYR1. Two missense mutations were previously reported in CCD patients. Clinically, patients with mutations in RYR1 showed milder phenotype compared with those without mutations. CONCLUSION: Congenital neuromuscular disease with uniform type 1 fiber (CNMDU1) in 40% of patients is associated with mutations in the C-terminal domain of RYR1, suggesting that CNMDU1 is allelic to central core disease at least in some patients.

Primary collagen VI deficiency is the second most common congenital muscular dystrophy in Japan.

OBJECTIVES: To determine the frequency of primary collagen VI deficiency in congenital muscular dystrophy (CMD) in Japan and to establish the genotype-phenotype correlation. METHODS: We performed immunohistochemistry for collagen VI in muscles from 362 Japanese patients with CMD, and directly sequenced the three collagen VI genes, COL6A1, COL6A2, and COL6A3, in patients found to have collagen VI deficiency. RESULTS: In Japan, primary collagen VI deficiency accounts for 7.2% of congenital muscular deficiency. Among these patients, five had complete deficiency (CD) and 29 had sarcolemma-specific collagen VI deficiency (SSCD). We found two homozygous and three compound heterozygous mutations in COL6A2 and COL6A3 in all five patients with CD, and identified heterozygous missense mutations or in-frame small deletions in 21 patients with SSCD in the triple helical domain (THD) of COL6A1, COL6A2, and COL6A3. All mutations in SSCD were sporadic dominant. No genotype-phenotype correlation was seen. CONCLUSION: Primary collagen VI deficiency is the second most common CMD after Fukuyama type CMD in Japan. Dominant mutations located in the N-terminal side from the cysteine residue in the THD of COL6A1, COL6A2, and COL6A3 are closely associated with SSCD.

Reduced cell anchorage may cause sarcolemma-specific collagen VI deficiency in Ullrich disease.

BACKGROUND: COL6 gene mutations are associated with Ullrich congenital muscular dystrophy (UCMD), which is clinically characterized by muscle weakness from early infancy, hyperlaxity of distal joints, and multiple proximal joint contractures. We previously reported that the majority of patients with UCMD have sarcolemma-specific collagen VI deficiency (SSCD). More recently, we found heterozygous COL6A1 glycine substitutions in patients with UCMD with SSCD. OBJECTIVE: To elucidate how COL6A1 glycine mutation leads to SSCD. METHODS: We evaluated the synthesis, formation, and binding of collagen VI to the extracellular matrix in fibroblasts with p.G284R mutation in COL6A1. RESULTS: Collagen VI was normally secreted into the cultured medium in fibroblasts harboring p.G284R mutation. When the medium with normal collagen VI was added to collagen VI-deficient fibroblast culture, collagen VI bound surrounding the cells, while collagen VI with p.G284R mutation did not. Cell adhesion of fibroblasts with p.G284R mutation was markedly reduced similarly to that of collagen VI-deficient cells. Interestingly, this reduction in adhesion of the cells with p.G284R mutation was recovered by the addition of the medium with normal collagen VI, which would suggest a therapeutic strategy for a replacement therapy. CONCLUSION: Heterozygous glycine substitution in COL6A1 may cause decreased binding of collagen VI microfibrils to the extracellular matrix resulting in sarcolemma-specific collagen VI deficiency.

Emerinopathy and laminopathy clinical, pathological and molecular features of muscular dystrophy with nuclear envelopathy in Japan.

Mutations in the genes for nuclear envelope proteins of emerin (EMD) and lamin A/C (LMNA) are known to cause Emery-Dreifuss muscular dystrophy (EDMD) and limb girdle muscular dystrophy (LGMD). We compared clinical features of the muscular dystrophy patients associated with mutations in EMD (emerinopathy) and LMNA (laminopathy) in our series. The incidence of laminopathy was slightly higher than that of emerinopathy. The age at onset of the disease in emerinopathy was variable and significantly older than in laminopathy. The initial symptom of emerinopathy was also variable, whereas nearly all laminopathy patients presented initially with muscle weakness. Calf hypertrophy was often seen in laminopathy, underscoring the importance of mutation screening for LMNA in childhood muscular dystrophy with calf hypertrophy. The clinical spectrum of emerinopathy is actually wider than previously known including EDMD, LGMD, conduction defects with minimal muscle/joint involvement, and their intermittent forms. Pathologically, no marked difference was observed between emerinopathy and laminopathy. Increased number and variation in size of myonuclei were detected. More precise observations using electron microscopy is warranted to characterize the detailed nuclear changes in nuclear envelopathy.

A new congenital form of X-linked autophagic vacuolar myopathy.

In a new family with X-linked congenital autophagic vacuolar myopathy (AVM), seven affected boys presented with congenital hypotonia, dyspnea, and dysphagia with delayed motor milestones. Muscle pathology revealed autophagic vacuoles with sarcolemmal features, multilayered basal lamina with marked sarcolemmal deposition of C5-9 membrane attack complex and calcium, histologically indistinguishable from childhood-onset X-linked myopathy with excessive autophagy (XMEA). Haplotype analysis suggests that this new AVM and XMEA may be allelic despite different clinical presentations.

Molecular pathomechanism of distal myopathy with rimmed vacuoles.

Distal myopathy with rimmed vacuoles (DMRV) and hereditary inclusion body myopathy (HIBM) are now known to be the same disease and are caused by mutations in tile GNE gene that encodes a bifunctional protein with two enzymatic activities: UDP-GlcNAc2-epimerase (GNE) and ManNAc kinase (MNK). GNE catalyzes the rate-limiting step in the sialic acid biosynthesis and MNK catalyzes the next step. So far, we have found homozygous or compound heterozygous mutations in 55 unrelated Japanese DMRV patients. Among them, c.1714G>C (p.V572L) mutation is the most common, accounting for 57% of the mutant alleles. The same mutation was recently identified also in Korean DMRV patients, raising the possibility of the presence of a common founder. We have also found that cardiac involvement is not very rare and is found in 18% of patients, albeit degree of severity widely varies; in some patients, it can result in sudden death. The length of time when patients become non ambulatory is diverse. The severity of clinical symptoms also varies widely, as evidenced by the presence of an asymptomatic homozygote harboring of p.D176V, the second most common mutation among Japanese patients. Patients' fibroblasts and myotubes are hyposialylated and this hyposialylation can be recovered by adding GNE metabolite, ManNAc, or sialic acid per se, NeuAc. Accordingly, the sialylation status in the skeletal muscle tissue is also greatly altered especially in fibers with rimmed vacuoles, suggesting the tight association between hyposialylation and the formation of rimmed vacuoles. However, we still do not know why hyposialylation leads to the formation of rimmed vacuoles. To further elucidate the pathomechanism and to develop a therapy of DMRV, we need to produce mouse model mouse for this disease.

X-linked form of Emery-Dreifuss muscular dystrophy.

Emery-Dreifuss muscular dystrophy (EDMD) is an inherited muscular disorder clinically characterized by slowly progressive weakness affecting humero-peroneal muscles, early joint contractures and cardiomyopathy with conduction defects. Autosomal dominant and recessive forms are caused by mutations in lamin A/C gene. Lamin A/C is a major component of nuclear lamina, and its gene mutations cause several human disorders including muscular dystrophy, cardiomyopathy, lipodystrophy, neuropathy, and progeria syndrome. X-linked recessive form of EDMD is caused by mutation in EMD (or STA) gene encoding an integral protein of the inner nuclear membrane. Emerin expresses ubiquitously, but its deficiency affects only limited tissues of skeletal and cardiac muscles and joints. In this paper, I will focus on clinical and pathological aspects of X-EDMD and possible functions of emerin.

Dysferlin mutation analysis in a group of Italian patients with limb-girdle muscular dystrophy and Miyoshi myopathy.

Mutations in the dysferlin gene (DYSF) on chromosome 2p13 cause distinct phenotypes of muscular dystrophy: limb-girdle muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy (MM), and distal anterior compartment myopathy, which are known by the term 'dysferlinopathy'. We performed mutation analyses of DYSF in 14 Italian patients from 10 unrelated families with a deficiency of dysferlin protein below 20% of the value in normal controls by immunoblotting analysis. We identified 11 different mutations, including eight missense and three deletion mutations. Nine of them were novel mutations. We also identified a unique 6-bp insertion polymorphism within the coding region of DYSF in 15% of Italian population, which was not observed in East Asian populations. The correlation between clinical phenotype and the gene mutations was unclear, which suggested the role of additional genetic and epigenetic factors in modifying clinical symptoms.

POMT1 mutation results in defective glycosylation and loss of laminin-binding activity in alpha-DG.

Walker-Warburg syndrome (WWS) is a congenital muscular dystrophy associated with neuronal migration disorder and structural eye abnormalities. The mutations in the O-mannosyltransferase 1 gene (POMT1) were identified recently in 20% of patients with WWS. The authors report on a patient with WWS and a novel POMT1 mutation. Their patient expressed alpha-dystroglycan (alpha-DG) core protein, but fully glycosylated alpha-DG antibody epitopes were absent, associated with the loss of laminin-binding activity.

Ullrich disease due to deficiency of collagen VI in the sarcolemma.

The authors identified eight patients with Ullrich disease in whom collagen VI was present in the interstitium but was absent from the sarcolemma. By electron microscopy, collagen VI in the interstitium was never linked to the basal lamina. These findings suggest that in these patients it is not the total absence of collagen VI from the muscle but the failure of collagen VI to anchor the basal lamina to the interstitium that is the cause of Ullrich disease. Only one of the patients had a mutation in the collagen VI gene, suggesting that the primary abnormality in most of the patients involved some other molecules.

Beyond LGMD1A: myotilin is a component of central core lesions and nemaline rods.

Myotilin is a Z-disc protein that binds alpha-actinin, gamma-filamin and F-actin. The essential role of myotilin in skeletal muscle is highlighted by the recent observation that autosomal dominant limb girdle muscular dystrophy type 1A is caused by mutations in the human myotilin gene. We studied the expression and subcellular distribution of myotilin in nemaline myopathy, central core disease, centronuclear myopathy, and myopathies with tubular aggregates. A prominent myotilin immunostaining of nemaline rods and core lesions was detected in all ten cases of nemaline myopathy and five cases of central core disease. This renders myotilin a sensitive, though non-specific marker for these structural lesions. Western blot analysis did not indicate an increased myotilin expression in nemaline myopathy muscle. However, the analysis indicated upregulation of a 75 kDa immunoreactive band, very weak in control muscle but previously detected in limb girdle muscular dystrophy 1A samples. Our findings indicate that myotilin is a core structural molecule in nemaline rods and central core lesions and suggest modification of myotilin in nemaline myopathy, and further support the notion that myotilin may have a key role in the dynamic molecular events mediating myofibril assembly in normal and diseased human skeletal muscle.

Fukutin-related protein gene mutated in the original kindred limb-girdle MD 2I.

The authors mapped an autosomal recessive form of limb-girdle MD on chromosome 19q13.3 (LGMD2I), further narrowed down the candidate region to 1.1 Mb, and identified one new homozygous mutation in the fukutin-related protein (FKRP) gene on patients of the original Tunisian family. Immunohistochemical and immunoblot analysis showed abnormal expression of alpha-dystroglycan and laminin-alpha2 supporting the hypothesis that FKRP has a role in the interaction between the extracellular matrix components.

Mutation in the caveolin-3 gene causes a peculiar form of distal myopathy.

The authors describe a patient with sporadic distal myopathy associated with reduced caveolin-3 in muscle fibers in which the muscle atrophy was restricted to the small muscles of the hands and feet. Gene analysis disclosed a heterozygous 80 G-->A substitution in the caveolin-3 gene that was identical to that of reported cases of elevated serum creatine kinase. This patient further demonstrated possible clinical heterogeneity of myopathies with mutations in the caveolin-3 gene.