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Distal myopathy with rimmed vacuoles or hereditary inclusion body myopathy is an adult-onset autosomal recessive, slowly progressive and debilitating myopathy due to mutations in the gene that regulates the synthesis of sialic acid. This review aims to update our knowledge of this myopathy and to review studies about pathomechanism and therapeutic strategies.Owing to the mutated gene, it was expected that the pathomechanism of this myopathy would be based on hyposialylation, a highly controversial phenomenon. This concept has been supported by findings in two recently generated animal models. In addition, the intracellular amyloid-beta accumulation in a distal myopathy with rimmed vacuole mouse model is relevant to similar findings in patients.Clarifying the role of hyposialylation in distal myopathy with rimmed vacuole/hereditary inclusion body myopathy could potentially lead to a therapeutic strategy for this progressive myopathy. In addition, strategies aimed at preventing amyloid-beta deposition or enhancing its clearance could also be beneficial, as this epiphenomenon is now known to occur early in the course of the disease.

Distal myopathy with rimmed vacuoles (DMRV), also called hereditary inclusion body myopathy (hIBM), is characterized clinically by weakness and atrophy that initially involves the distal muscles and pathologically by the presence of rimmed vacuoles (RVs) or intracellular protein deposits in myofibers. It is caused by mutations in the UDP- N-acetylglucosamine 2-epimerase/ N-acetylmannosamine kinase ( GNE) gene that is important in sialic acid synthesis. Recently, we generated a mouse model ( Gne−/−h GNED176VTg) that exhibits muscle weakness and pathological changes similar to DMRV patients. To gain better understanding of the pathomechanism of DMRV, we determined temporal changes in the overall motor performance of this model mouse for DMRV in correlation with the structure and function of isolated skeletal muscles and muscle pathology. These DMRV mice exhibited muscle weakness, decreased whole muscle mass and cross-sectional area (CSA), and reduced contractile power in an age-related manner. Single-fiber CSA further supported the finding of muscle atrophy that involved both type I and type II fibers. These results suggest that atrophy is highly correlated with reduced production of force at young age, both in vivo and ex vivo, thereby implicating the important role of atrophy in the pathomechanism of DMRV. In older age, and particularly in gastrocnemius muscles, RVs and intracellular inclusions were seen in type IIA fibers, further aggravating reduction of force and specific increase in twitch-tetanus ratio.

Distal myopathy is a group of heterogeneous disorders affecting predominantly distal muscles usually appearing from young to late adulthood with very rare cardiac complications. We report a 27-year-old man characterized clinically by distal myopathy and dilated cardiomyopathy, pathologically by lipid storage, and genetically by a PNPLA2 mutation. The patient developed weakness in his lower legs and fingers at age 20 years. Physical examination at age 27 years revealed muscle weakness and atrophy predominantly in lower legs and hands, and severe dilated cardiomyopathy. The patient had a homozygous four-base duplication (c.475_478dupCTCC) in exon 4 of PNPLA2.

Background In chicken, loss of TALPID3 results in non-functional cilia and short-rib polydactyly syndrome. This phenotype is caused by a frameshift mutation in the chicken ortholog of the human KIAA0586 gene, which encodes a novel coiled-coil domain protein essential for primary ciliogenesis, suggesting that KIAA0586 can be associated with ciliopathy in human beings. Methods In our patients with ciliopathy (http://www.clinicaltrials.gov: NCT00068224), we have collected extensive clinical and neuroimaging data from affected individuals, and performed whole exome sequencing on DNA from affected individuals and their parents. We analysed gene expression on fibroblast cell line, and determined the effect of gene mutation on ciliogenesis in cells derived from patients. Results We identified biallelic mutations in the human TALPID3 ortholog, KIAA0586 , in six children with findings of overlapping Jeune and Joubert syndromes. Fibroblasts cultured from one of the patients with Jeune–Joubert syndrome exhibited more severe cilia defects than fibroblasts from patients with only Joubert syndrome; this difference was reflected in KIAA0586 RNA expression levels. Rescue of the cilia defect with full-length wild type KIAA0586 indicated a causal link between cilia formation and KIAA0586 function. Conclusions Our results show that biallelic deleterious mutations in KIAA0586 lead to Joubert syndrome with or without Jeune asphyxiating thoracic dystrophy. Furthermore, our results confirm that KIAA0586/TALPID3 is essential in cilia formation in human beings, expand the KIAA0586 phenotype to include features of Jeune syndrome and provide a pathogenetic connection between Joubert and Jeune syndromes, based on aberrant ciliogenesis.

Background Laminins are heterotrimeric complexes, consisting of α, β and γ subunits that form a major component of basement membranes and extracellular matrix. Laminin complexes have different, but often overlapping, distributions and functions. Methods Under our clinical protocol, NCT00068224, we have performed extensive clinical and neuropsychiatric phenotyping, neuroimaging and molecular analysis in patients with laminin α1 ( LAMA1 )-associated lamininopathy. We investigated the consequence of mutations in LAMA1 using patient-derived fibroblasts and neuronal cells derived from neuronal stem cells. Results In this paper we describe individuals with biallelic mutations in LAMA1 , all of whom had the cerebellar dysplasia, myopia and retinal dystrophy, in addition to obsessive compulsive traits, tics and anxiety. Patient-derived fibroblasts have impaired adhesion, reduced migration, abnormal morphology and increased apoptosis due to impaired activation of Cdc42, a member of the Rho family of GTPases that is involved in cytoskeletal dynamics. LAMA1 knockdown in human neuronal cells also showed abnormal morphology and filopodia formation, supporting the importance of LAMA1 in neuronal migration, and marking these cells potentially useful tools for disease modelling and therapeutic target discovery. Conclusion This paper broadens the phenotypes associated with LAMA1 mutations. We demonstrate that LAMA1 deficiency can lead to alteration in cytoskeletal dynamics, which may invariably lead to alteration in dendrite growth and axonal formation. Estimation of disease prevalence based on population studies in LAMA1 reveals a prevalence of 1–20 in 1 000 000. Trial registration number: NCT00068224

Ryanodine receptor 1 ( RYR1 ) gene mutations are associated with central core disease (CCD), multiminicore disease (MmD) and malignant hyperthermia (MH), and have been reported to be responsible for 47–67% of patients with CCD and rare cases with MmD. However, to date, the true frequency and distribution of the mutations along the RYR1 gene have not been determined yet, since mutation screening has been limited to three ‘hot spots’, with particular attention to the C-terminal region. In this study, 27 unrelated Japanese CCD patients were included. Clinical histories and muscle biopsies were carefully reviewed. We sequenced all the 106 exons encoding RYR1 with their flanking exon–intron boundaries, and identified 20 novel and 3 previously reported heterozygous missense mutations in 25 of the 27 CCD patients (93%), which is a much higher mutation detection rate than that perceived previously. Among them, six were located outside the known ‘hot spots’. Sixteen of 27 (59%) CCD patients had mutations in the C-terminal ‘hot spot’. Three CCD patients had a probable autosomal recessive disease with two heterozygous mutations. Patients with C-terminal mutations had earlier onset and rather consistent muscle pathology characterized by the presence of distinct cores in almost all type 1 fibres, interstitial fibrosis and type 2 fibre deficiency. In contrast, patients with mutations outside the C-terminal region had milder clinical phenotype and harbour more atypical cores in their muscle fibres. We also sequenced two genes encoding RYR1-associated proteins as candidate causative genes for CCD: the 12 kD FK506-binding protein ( FKBP12 ) and the α1 subunit of L-type voltage-dependent calcium channel or dihydropyridine receptor ( CACNA1S ). However, no mutation was found, suggesting that these genes may not, or only rarely, be responsible for CCD. Our results indicate that CCD may be caused by RYR1 mutations in the majority of patients.

Induced pluripotent stem cells (iPSCs) are an essential tool for modeling how causal genetic variants impact cellular function in disease, as well as an emerging source of tissue for regenerative medicine. The preparation of somatic cells, their reprogramming and the subsequent verification of iPSC pluripotency are laborious, manual processes limiting the scale and reproducibility of this technology. Here we describe a modular, robotic platform for iPSC reprogramming enabling automated, high-throughput conversion of skin biopsies into iPSCs and differentiated cells with minimal manual intervention. We demonstrate that automated reprogramming and the pooled selection of polyclonal pluripotent cells results in high-quality, stable iPSCs. These lines display less line-to-line variation than either manually produced lines or lines produced through automation followed by single-colony subcloning. The robotic platform we describe will enable the application of iPSCs to population-scale biomedical problems including the study of complex genetic diseases and the development of personalized medicines.

The GNE gene encodes the rate-limiting, bifunctional enzyme of sialic acid biosynthesis, uridine diphosphate-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). Biallelic GNE mutations underlie GNE myopathy, an adult-onset progressive myopathy. GNE myopathy-associated GNE mutations are predominantly missense, resulting in reduced, but not absent, GNE enzyme activities. The exact pathomechanism of GNE myopathy remains unknown, but likely involves aberrant (muscle) sialylation. Here, we summarize 154 reported and novel GNE variants associated with GNE myopathy, including 122 missense, 11 nonsense, 14 insertion/deletions, and seven intronic variants. All variants were deposited in the online GNE variation database (http://www.dmd.nl/nmdb2/home.php?select_db=GNE). We report the predicted effects on protein function of all variants well as the predicted effects on epimerase and/or kinase enzymatic activities of selected variants. By analyzing exome sequence databases, we identified three frequently occurring, unreported GNE missense variants/polymorphisms, important for future sequence interpretations. Based on allele frequencies, we estimate the world-wide prevalence of GNE myopathy to be ∼4-21/1,000,000. This previously unrecognized high prevalence confirms suspicions that many patients may escape diagnosis. Awareness among physicians for GNE myopathy is essential for the identification of new patients, which is required for better understanding of the disorder's pathomechanism and for the success of ongoing treatment trials.

Distal myopathy with rimmed vacuoles (DMRV) is an autosomal recessive disorder characterised by early adultonset (15–40 years), slowly progressive myopathy with preferential weakness of the tibialis anterior and sparing of the quadriceps femoris muscles [1]. The muscle biopsy shows rimmed vacuoles containing deposits immunoreactive for b-amyloid (Ab) and other proteins. DMRV is due to mutations in the GNE gene, which encodes a bifunctional enzyme (uridine diphosphate-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase) in the sialic acid synthetic pathway. A recently developed mouse model of DMRV that expresses the human GNE D176V mutation in a Gne knockout background reproduces the clinical, pathological and biochemical features of human DMRV [2]. As in the human disease the model exhibited muscle atrophy and weakness, and low levels of sialic acid in serum and solid organs. Moreover, Abwas also found to be associated with the rimmed vacuoles and myofibrillar degeneration in the muscle fibres [2]. Because Alzheimer’s disease (AD) is characterised by Ab deposition in the brain, we examined the central nervous system of the DMRV mouse model for evidence of Ab deposition and other pathological abnormalities to determine if there is any link between DMRV and AD. Brain and skeletal muscle tissues from 30 DMRV mice older than 40 weeks were harvested, formalin-fixed, processed and paraffin embedded. Sixteen spinal cords were also available for examination. Tissues sections were stained by hematoxylin and eosin for light microscopy. Immunohistochemistry (IHC) to detect Ab was performed using a mouse monoclonal primary antibody (clone 6E/10; Covance, Princeton, NJ) and the Envision method with minor modifications [3]. As a positive IHC control, we used tissues from an established mouse model of AD, the Tg2576 transgenic mouse (Taconic, Germantown, NY) that expresses the Swedish mutation of amyloid precursor protein. Brain tissue sections from a human case of AD were also included as a positive control. For negative controls, sections were incubated with normal goat serum to replace the primary antibody. In the 30 aged DMRV mice examined there was no evidence of Ab deposits in the brains or spinal cords (Figure 1B). However, Ab deposits were detected in the skeletal muscles of all these animals. The deposits were usually linear, beaded and found in the central part of the fibres (Figure 1C, D). Occasionally, these deposits were associated with vacuoles (Figure 1C) corresponding to rimmed vacuoles as shown by the hematoxylin and eosin stains (data not shown). The three AD mouse brain positive controls showed plaque-like amyloid deposits in the cerebral cortex (Figure 1A). The human AD brain tissues showed Ab deposition in the brain neuropil and around blood vessels (data not shown). There were no light microscopic abnormalities such as neurofibrillary tangles, amyloid angiopathy and other features of AD in the central nervous system of all test and control mice. Although the aged DMRV mouse model demonstrated convincing Ab deposition in skeletal muscles, there was no Ab deposition in the CNS. This suggests that amyloidogenesis in AD and DMRV may be different. Amyloidogenesis in AD is mainly associated with post-translational proteolytic processing of amyloid precursor protein by a-, band g-secretases. However, hyposialylation is an important factor in the pathogenesis of the disease and amyloidogenesis in DMRV. Hyposialylation of several important muscle glycoproteins Keywords

Ullrich congenital muscular dystrophy (UCMD) is an inherited muscle disorder characterized clinically by muscle weakness, distal joint hyperlaxity, and proximal joint contractures. Sporadic and recessive mutations in the three collagen VI genes, COL6A1, COL6A2, and COL6A3, are reported to be causative. In the sporadic forms, a heterozygous point mutation causing glycine substitution in the triple helical domain has been identified in higher rate. In this study, we examined the efficacy of siRNAs, which target point mutation site, on specific knockdown toward transcripts from mutant allele and evaluated consequent cellular phenotype of UCMD fibroblasts. We evaluated the effect of siRNAs targeted to silence-specific COL6A1 alleles in UCMD fibroblasts, where simultaneous expression of both wild-type and mutant collagen VI resulted in defective collagen localization. Addition of mutant-specific siRNAs allowed normal extracellular localization of collagen VI surrounding fibroblasts, suggesting selective inhibition of mutant collagen VI. Targeting the single-nucleotide COL6A1 c.850G>A (p.G284R) mutation responsible a sporadic autosomal dominant form of UCMD can potently and selectively block expression of mutant collagen VI. These results suggest that allele-specific knockdown of the mutant mRNA can potentially be considered as a therapeutic procedure in UCMD due to COL6A1 point mutations.

Summary Background and objectives Elevated serum vitamin D with hypercalciuria can result in nephrocalcinosis and nephrolithiasis. This study evaluated the cause of excess 1,25-dihydroxycholecalciferol (1α,25(OH)2D3) in the development of those disorders in two individuals. Design, setting, participants, & measurements Two patients with elevated vitamin D levels and nephrocalcinosis or nephrolithiasis were investigated at the National Institutes of Health (NIH) Clinical Center and the NIH Undiagnosed Diseases Program, by measuring calcium, phosphate, and vitamin D metabolites, and by performing CYP24A1 mutation analysis. Results Both patients exhibited hypercalciuria, hypercalcemia, low parathyroid hormone, elevated vitamin D (1α,25(OH)2D3), normal 25-OHD3, decreased 24,25(OH)2D, and undetectable activity of 1,25(OH)2D-24-hydroxylase (CYP24A1), the enzyme that inactivates 1α,25(OH)2D3. Both patients had bi-allelic mutations in CYP24A1 leading to loss of function of this enzyme. On the basis of dbSNP data, the frequency of predicted deleterious bi-allelic CYP24A1 variants in the general population is estimated to be as high as 4%–20%. Conclusions The results of this study show that 1,25(OH)2D-24-hydroxylase deficiency due to bi-allelic mutations in CYP24A1 causes elevated serum vitamin D, hypercalciuria, nephrocalcinosis, and renal stones.

Sialic acids are terminal sugars of glycolipids and glycoproteins and are involved in several cellular processes. Sialic acid biosynthesis occurs in the cytosol, where UDP-N-acetylglucosamine (GlcNAc) is sequentially converted to N-acetylmannosamine (ManNAc) 6-phosphate by UDP-GlcNAc-2-epimerase/ManNAc kinase enzymes, both of which are encoded by the GNE gene. Since the only existing mouse model of DMRV/hIBM (Gne(-/-)hGNED176VTg) exhibited decreased sialic acid levels in most organs, DMRV/hIBM is thought to be secondary to the metabolic defect in sialic acid production. Theoretically, replenishing sialic acid could be employed as a therapeutic option. It has been reported that N-acetylneuraminic acid (NeuAc) and ManNAc are well incorporated into cells and converted to sialic acid. Thus, we evaluated the efficacy and safety of ManNAc, NeuAc, and sialyllactose in the Gne(-/-)hGNED176VTg, by orally administering these agents to mice from 5-15 weeks continuously until they reached 54-57 weeks of age. The treatment showed beneficial effects in terms of survival rate, overall motor performance, myofiber size, ex vivo skeletal muscle contractile properties, and pathology. These low-dose compounds showed acceptable kidney and liver toxicity profiles. Thus our results show that the oral therapy with NeuAc and ManNAc or their derivatives is safe and effective in preventing myopathic symptoms in Gne(-/-)hGNED176VTg mice, and could be considered as a guide for further therapeutic trials.

Distal myopathy with rimmed vacuoles (DMRV), also called hereditary inclusion body myopathy, is an autosomal recessive disorder caused by homozygous or compound heterozygous missense mutations in GNE which encodes a protein with two enzymatic activities in sialic acid biosynthesis: UDP-GlcNAc 2-epimerase and ManNAc kinase. The disease starts from 1540 years and is slowly progressive. DMRV preferentially affects tibialis anterior and hamstrings muscles, and has characteristic findings in muscle pathology which include rimmed vacuoles, tubulofilamentous inclusions, deposition of amyloid, and phosphorylated tau. We generated DMRV mice (Gne -/- hGNE D176V-Tg) by crossmating Gne knock-out heterozygous mouse and human GNE p.D176V transgenic mouse. This model mouse recapitulates DMRV clinically, pathologically, and biochemically by developing muscle weakness and atrophy from 21 weeks, amyloid deposition from 31 weeks, and rimmed vacuoles and phosphorylated tau from 41 weeks while having lifelong hyposialylation. We gave three types of GNE metabolites, ManNAc, NeuAc and sialyllactose, to DMRV mice orally from 15 weeks until 55 weeks of age. Sialic acid supplementation almost completely precluded the disease and virtually no sign of DMRV was seen even at 55 weeks of age, indicating that decreased sialic acid is the cause of myopathic phenotype and sialic acid supplementation can prevent the disease process.

Autophagy is a tightly regulated pathway for the degradation and recycling of proteins delivered to lysosomes, and is an important process in maintaining cellular homeostasis. Whereas a basal level of autophagy can be detected in skeletal muscles, its perturbation can be seen in a variety of conditions affecting the muscle. In certain muscle diseases, moreover, autophagy seems to be a characteristic feature, although the exact role of autophagy in these disorders is just starting to be understood. As autophagy is not only an index of disease progression but also a potential target for treatment in certain disease conditions, its characterization is indeed of relevance. Thus, in this chapter, methods applicable to both human and murine skeletal muscle preparation for the analysis and monitoring of autophagy are presented.

Autophagy is a tightly regulated pathway for the degradation and recycling of proteins delivered to lysosomes, and is an important process in maintaining cellular homeostasis. Whereas a basal level of autophagy can be detected in skeletal muscles, its perturbation can be seen in a variety of conditions affecting the muscle. In certain muscle diseases, moreover, autophagy seems to be a characteristic feature, although the exact role of autophagy in these disorders is just starting to be understood. As autophagy is not only an index of disease progression but also a potential target for treatment in certain disease conditions, its characterization is indeed of relevance. Thus, in this chapter, methods applicable to both human and murine skeletal muscle preparation for the analysis and monitoring of autophagy are presented.

Distal myopathy with rimmed vacuoles (DMRV)-hereditary inclusion body myopathy (hIBM) is an adult-onset, moderately progressive autosomal recessive myopathy; eventually, affected individuals become wheelchair bound1. It is characterized clinically by skeletal muscle atrophy and weakness, and pathologically by rimmed vacuoles, which are actually accumulations of autophagic vacuoles2, 3, 4, scattered angular fibers and intracellular accumulation of amyloid and other proteins5. To date, no therapy is available for this debilitating myopathy, primarily because the disease pathomechanism has been enigmatic. It is known that the disease gene underlying DMRV-hIBM is GNE, encoding glucosamine (UDP-N-acetyl)-2-epimerase and N-acetylmannosamine kinase6, 7, 8--two essential enzymes in sialic acid biosynthesis9. It is still unclear, however, whether decreased sialic acid production causes muscle degeneration, as GNE has been proposed to have roles other than for sialic acid biosynthesis10, 11, 12. By showing that muscle atrophy and weakness are completely prevented in a mouse model of DMRV-hIBM after treatment with sialic acid metabolites orally, we provide evidence that hyposialylation is indeed one of the key factors in the pathomechanism of DMRV-hIBM. These results support the notion that DMRV-hIBM can potentially be treated simply by giving sialic acids, a strategy that could be applied in clinical trials in the near future.

Distal myopathies are a group of heterogeneous disorders classified into one broad category due to the presentation of weakness involving the distal skeletal muscles. The recent years have witnessed increasing efforts to identify the causative genes for distal myopathies. The identification of few causative genes made the broad classification of these diseases under "distal myopathies" disputable and added some enigma to why distal muscles are preferentially affected. Nevertheless, with the clarification of the molecular basis of specific conditions, additional clues have been uncovered to understand the mechanism of each condition. This review will give a synopsis of the common distal myopathies, presenting salient facts regarding the clinical, pathological, and molecular aspects of each disease. Distal myopathy with rimmed vacuoles, or Nonaka myopathy, will be discussed in more detail.

In Ullrich congenital muscular dystrophy, due to heterozygous mutations in COL6 genes, collagen VI is preserved in the interstitium but lost in the sarcolemma. We found that the binding ability of mutated collagen VI to extracellular matrix was markedly reduced compared to control. This indicates that heterozygous mutations in COL6 genes diminish the anchorage of collagen VI microfibrils to the extracellular matrix surrounding myocytes. This is the cause for sarcolemma-specific collagen VI deficiency.

Lysosomes are membrane-bound acidic organelles that contain hydrolases used for intracellular digestion of various macromolecules in a process generally referred to as autophagy. In normal skeletal and cardiac muscles, lysosomes usually appear morphologically unremarkable and thus are not readily visible on light microscopy. In distinct neuromuscular disorders, however, lysosomes have been shown to be structurally abnormal and functionally impaired, leading to the accumulation of autophagic vacuoles in myofibers. More specifically, there are myopathies in which buildup of these autophagic vacuoles seem to predominate the pathological picture. In such conditions, autophagy is considered not merely a secondary event, but a phenomenon that actually contributes to disease pathomechanism and/or progression. At present, there are two disorders in the muscle which are associated with primary defect in lysosomal proteins, namely Danon disease and Pompe disease. Other myopathies which have prominent autophagy in the skeletal muscle include X-linked myopathy with excessive autophagy (XMEA). In this review, these disorders are briefly characterized, and the role of autophagy in the context of the pathomechanism of these disorders is highlighted.

Distal myopathy with rimmed vacuoles (DMRV) or hereditary inclusion myopathy (h-IBM) is an early adult-onset distal myopathy caused by mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene which encodes for a bifunctional enzyme involved in sialic acid biosynthesis. It is pathologically characterized by the presence of rimmed vacuoles especially in atrophic fibers, which also occasionally contain congophilic materials that are immunoreactive to beta-amyloid, lysosomal proteins, ubiquitin and tau proteins. To elucidate the pathomechanism of this myopathy and to explore the treatment options, we generated a mouse model of DMRV/h-IBM. We knocked out the Gne gene in the mouse, but this resulted in embryonic lethality. We therefore generated a transgenic mouse that expressed the human GNEV572L mutation, which is the most prevalent among Japanese DMRV patients, and crossed this with Gne((+/-)) mouse to obtain Gne((-/-))hGNEV572L-Tg. Interestingly, these mice exhibit marked hyposialylation in serum, muscle and other organs. Reduction in motor performance in these mice can only be seen from 30 weeks of age. A compelling finding is the development of beta-amyloid deposition in myofibers by 32 weeks, which clearly precedes rimmed vacuole formation at 42 weeks. These results show that the Gne((-/-)) hGNEV572L-Tg mouse mimics the clinical, histopathological and biochemical features of DMRV/h-IBM, making it useful for understanding the pathomechanism of this myopathy and for employing different strategies for therapy. Our findings underscore the notion that hyposialylation plays an important role in the pathomechanism of DMRV/h-IBM.

Distal myopathy with rimmed vacuoles (DMRV) or hereditary inclusion myopathy (h-IBM) is an early adult-onset distal myopathy caused by mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene which encodes for a bifunctional enzyme involved in sialic acid biosynthesis. It is pathologically characterized by the presence of rimmed vacuoles especially in atrophic fibers, which also occasionally contain congophilic materials that are immunoreactive to beta-amyloid, lysosomal proteins, ubiquitin and tau proteins. To elucidate the pathomechanism of this myopathy and to explore the treatment options, we generated a mouse model of DMRV/h-IBM. We knocked out the Gne gene in the mouse, but this resulted in embryonic lethality. We therefore generated a transgenic mouse that expressed the human GNEV572L mutation, which is the most prevalent among Japanese DMRV patients, and crossed this with Gne((+/-)) mouse to obtain Gne((-/-))hGNEV572L-Tg. Interestingly, these mice exhibit marked hyposialylation in serum, muscle and other organs. Reduction in motor performance in these mice can only be seen from 30 weeks of age. A compelling finding is the development of beta-amyloid deposition in myofibers by 32 weeks, which clearly precedes rimmed vacuole formation at 42 weeks. These results show that the Gne((-/-)) hGNEV572L-Tg mouse mimics the clinical, histopathological and biochemical features of DMRV/h-IBM, making it useful for understanding the pathomechanism of this myopathy and for employing different strategies for therapy. Our findings underscore the notion that hyposialylation plays an important role in the pathomechanism of DMRV/h-IBM.

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.1714GC (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.

Distal myopathy with rimmed vacuoles (DMRV) and hereditary inclusion body myopathy (HIBM) are genetically identical autosomal recessive muscle disorders caused by mutations in the GNE gene. This gene encodes a bifunctional protein with UDP-GlcNAc 2-epimerase and ManNAc kinase activities that catalyze the rate limiting step and the succeeding step, respectively, in the sialic acid biosynthetic pathway. V572L mutation is the most prevalent among Japanese DMRV patients and accounts for about 60% of mutant alleles. Clinical spectrum of DMRV/HIBM seems to be wider than previously thought in terms of both the severity of the disease and the range of affected organs. There are rare asymptomatic homozygotes with missense GNE mutations, indicating the presence of mitigating factors. Surprisingly, more than 10% of the patients had a variety of cardiac abnormalities, suggesting that skeletal muscle may not be the only organ involved. Studies on recombinant GNE demonstrate a loss-of-function nature of the missense mutations identified. Patients' cells show decreased sialylation status which can be recovered by adding GNE metabolites, such as ManNAc and NeuAc. This indicates the possibility of developing a therapy for DMRV/HIBM by giving these metabolites to patients although we have to await the model mice that are currently being produced at several laboratories.

GNE myopathy is an adult-onset distal myopathy caused by mutations in the GNE gene, which codes for a bifunctional enzyme important in sialic acid biosynthesis. GNE myopathy is characterized by gradually progressive weakness and atrophy that preferentially involves distal extremities. Muscle degeneration occurs, with accumulation of inclusion bodies and rimmed vacuoles in muscle fibers. Although GNE myopathy is known to preferential affect skeletal muscles, there had been a few reports demonstrating involvement of the cardiac muscles. Through our Natural History Study (NCT01417533), we found that cardiac involvement in GNE myopathy patients may have a higher occurrence than expected. In this study we established a systematic evaluation of the cardiac involvement in GNE myopathy by analyzing the mouse model Gne-/-hGNED176VTg. Histopathology showed the presence of rimmed vacuoles and disorganization of cardiac myofibrils. Lectin staining array corroborated the hyposialylation of O-linked glycoproteins. Echocardiogram revealed decreased ejection fraction and fractional shortening, and increased left ventricle mass, indicating a decrease of cardiac function. These data were also confirmed by functional MRI on the mouse model. Our findings provide evidence that cardiac muscles are involved in GNE myopathy. We propose that hyposialylation of cardiac muscles can lead to impaired cardiac muscle contractility, and may be improved with sialylation-increasing therapies. It is important for clinicians to be aware of the possible occurrence of cardiac disease in GNE myopathy for careful examination of the cardiac function in patients and proper management.

GNE myopathy (also known as distal myopathy with rimmed vacuoles or hereditary inclusion body myopathy) is a rare autosomal recessive myopathy characterized by skeletal muscle atrophy and weakness that preferentially involve the distal muscles. The causative GNE gene encodes an essential enzyme in the biosynthesis of sialic acid and hyposialylation is thought to be a key factor in the underlying pathomechanism. We previously found that muscle atrophy appears in the young age of GNE myopathy model mice and is preventable by antioxidant administration, suggesting that oxidative stress would be associated with this primary myopathic phenotype. To link hyposialylation with the pathologic features, we investigated cellular response to oxidative stress in GNE myopathy myotubes. Skeletal muscle myotubes were prepared from Gne −/− hGNED176VTg mice and littermate controls. Intracellular reactive oxygen species (ROS) and cell viability were evaluated. First, we found that intracellular ROS generation was more profound in GNE myopathy myotubes compared to littermates. Then the susceptibility of GNE myopathy myotubes to increasing concentrations of menadione, a generator of ROS, with manipulating cellular sialylation was analyzed. We found that hyposialylated myotubes cultured in serum-free media are more vulnerable to menadione-induced oxidative stress. Dose-dependent increase in intracellular oxidants and cell mortality were weakened with the addition of NeuAc to culture media, which implies that normalization of sialylation levels improved intracellular antioxidant capacities. Our results provide a clue how hyposialylation makes the pathognomonic change in GNE myopathy and suggest that oxidative stress can be a therapeutic target of this progressive disease.

Rimmed vacuoles in myofibers are thought to be due to the accumulation of autophagic vacuoles, and can be characteristic in certain myopathies with protein inclusions in myofibers. In this study, we performed a detailed clinical, molecular, and pathological characterization of Becker muscular dystrophy patients who have rimmed vacuoles in muscles. Among 65 Becker muscular dystrophy patients, we identified 12 patients who have rimmed vacuoles and 11 patients who have deletions in exons 45-48 in DMD gene. All patients having rimmed vacuoles showed milder clinical features compared to those without rimmed vacuoles. Interestingly, the rimmed vacuoles in Becker muscular dystrophy muscles seem to represent autophagic vacuoles and are also associated with polyubiquitinated protein aggregates. These findings support the notion that rimmed vacuoles can appear in Becker muscular dystrophy, and may be related to the chronic changes in muscle pathology induced by certain mutations in the DMD gene.

Ullrich congenital muscular dystrophy (UCMD) is an inherited muscle disorder characterized clinically by muscle weakness, distal joint hyperlaxity, and proximal joint contractures. Sporadic/dominant and recessive mutations in the three collagen VI genes, COL6A1 , COL6A2 , and COL6A3 , are reported to be causative. In the sporadic/dominant form, heterozygous point mutations causing glycine substitution in the triple helical domain have been identified. In this study, we examined the effects of mutant allele mRNA-specific knockdown using siRNAs that target point mutations in COL6A1 on the phenotype of UCMD fibroblasts. We designed several siRNAs for COL6A1 point mutation with/without a mismatched base and measured gene-knockdown effect and specificity of those siRNAs on murine cell-lines transfected with normal and mutant human COL6A1 cDNA. The selected siRNAs were introduced into patient’s fibroblasts, and then the cell-associated collagen VI expression and the ability for cell–substrate attachment were evaluated. Two siRNAs were selected out in transfection experiments. Both worked well on the recovery of extracellular localization of collagen VI surrounding fibroblasts, while on cell attachment assay, one of them showed remarkable recovery of cell-attachment. These results suggest that allele-specific knockdown of the mutant mRNA can potentially be considered a therapeutic strategy in UCMD due to COL6A1 point mutations.

GNE myopathy (GM), which is also known as distal myopathy with rimmed vacuoles or hereditary inclusion body myopathy, is an autosomal recessive myopathy characterized by skeletal muscle atrophy and weakness that preferentially involve the distal muscles. The histopathologic features in muscle biopsy include the myofiber atrophy with the presence of rimmed vacuoles and intracellular amyloid deposits. Although it has been already demonstrated that sialic acid treatment can prevent the development of myopathic phenotype in GM model mouse, there remains an unexplained link between hyposialylation due to GNE mutations and the pathognomonic findings in the muscle. Previously we have reported that muscle atrophy is one of the early signs of myopathic phenotype in the mouse model. In this study, we evaluated several phenomena that contribute to atrophy in muscles and focused on reactive oxygen species (ROS) production. We measured the levels of hydroxyl radicals after induced muscle contraction in vivo. We found profound hydroxyl radical increments in GM mice as compared to littermates, indicating a decreased antioxidative capacity in the GM muscles. The implication of oxidative stress in disease mechanism was further supported by the finding that several genes responsive to oxidative stress were highly expressed in the GNE myopathy muscles. The evidence that muscle atrophy is associated with oxidative stress was further reinforced by the improvement in muscle strength in GM mice given α-tocopherol, and the increase in myofiber diameter in GM mice treated with N-acetylcysteine. These results not only provide important clues on understanding GM pathomechanism but more importantly demonstrate the potential of antioxidants in therapeutic regimen of GM patients.

GNE myopathy (GM) is caused by mutations in GNE gene, encoding an essential enzyme in sialic acid biosynthesis. We previously provided evidence that sialic acid given to clinically asymptomatic GM mice prevented the development of the myopathic phenotype, indicating that hyposialylation is one of key factors in the pathomechanism underlying GM. In this study, we examined the effect of sialic acid administration on muscle atrophy and weakness in symptomatic, older GM mice to mimic the stage of disease in symptomatic GM patients, using free sialic acid (NeuAc) and the less-metabolized sialic acid conjugate, 6′-sialyllactose (6′SL). Fifty-week-old GM mice and littermates were given either water, NeuAc, or 6’SL for about 30 weeks. Voluntary exercise on running wheel was evaluated longitudinally at 10 week interval, while the size, force generation, and pathology of gastrocnemius muscle (GC) were evaluated at the end of the study. Voluntary exercise in non-treated model mice markedly decreased with aging, but was maintained in 6′SL group, including one mouse that showed marked recovery to non-affected control level. The NeuAc group, on the other hand, exhibited marked decrease in voluntary wheel running similar to non-treated mice. The response in running performance correlated with findings in analysis of GC, i.e., the GC size and force production in 6′SL group was recovered to levels measured in control littermates, while those in the NeuAc group responded poorly and showed similar findings with non-treated GM mice. We show that 6′SL can ameliorate muscle atrophy and weakness in symptomatic GM mice, providing a proof of principle in the use of this compound in the clinical trial of GM patients with progressive or advanced stage of disease.