Ichizo Nishino

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive human disease associated with multiple deletions of skeletal muscle mitochondrial DNA (mtDNA), which have been ascribed to a defect in communication between the nuclear and mitochondrial genomes. Examination of 12 MNGIE probands revealed homozygous or compound-heterozygous mutations in the gene specifying thymidine phosphorylase (TP), located on chromosome 22q13.32-qter. TP activity in leukocytes from MNGIE patients was less than 5 percent of controls, indicating that loss-of-function mutations in TP cause the disease. The pathogenic mechanism may be related to aberrant thymidine metabolism, leading to impaired replication or maintenance of mtDNA, or both.

FOXO1, a member of the FOXO forkhead type transcription factors, is markedly up-regulated in skeletal muscle in energy-deprived states such as fasting and severe diabetes, but its functions in skeletal muscle have remained poorly understood. In this study, we created transgenic mice specifically overexpressing FOXO1 in skeletal muscle. These mice weighed less than the wild-type control mice, had a reduced skeletal muscle mass, and the muscle was paler in color. Microarray analysis revealed that the expression of many genes related to the structural proteins of type I muscles (slow twitch, red muscle) was decreased. Histological analyses showed a marked decrease in size of both type I and type II fibers and a significant decrease in the number of type I fibers in the skeletal muscle of FOXO1 mice. Enhanced gene expression of a lysosomal proteinase, cathepsin L, which is known to be up-regulated during skeletal muscle atrophy, suggested increased protein degradation in the skeletal muscle of FOXO1 mice. Running wheel activity (spontaneous locomotive activity) was significantly reduced in FOXO1 mice compared with control mice. Moreover, the FOXO1 mice showed impaired glycemic control after oral glucose and intraperitoneal insulin administration. These results suggest that FOXO1 negatively regulates skeletal muscle mass and type I fiber gene expression and leads to impaired skeletal muscle function. Activation of FOXO1 may be involved in the pathogenesis of sarcopenia, the age-related decline in muscle mass in humans, which leads to obesity and diabetes. FOXO1, a member of the FOXO forkhead type transcription factors, is markedly up-regulated in skeletal muscle in energy-deprived states such as fasting and severe diabetes, but its functions in skeletal muscle have remained poorly understood. In this study, we created transgenic mice specifically overexpressing FOXO1 in skeletal muscle. These mice weighed less than the wild-type control mice, had a reduced skeletal muscle mass, and the muscle was paler in color. Microarray analysis revealed that the expression of many genes related to the structural proteins of type I muscles (slow twitch, red muscle) was decreased. Histological analyses showed a marked decrease in size of both type I and type II fibers and a significant decrease in the number of type I fibers in the skeletal muscle of FOXO1 mice. Enhanced gene expression of a lysosomal proteinase, cathepsin L, which is known to be up-regulated during skeletal muscle atrophy, suggested increased protein degradation in the skeletal muscle of FOXO1 mice. Running wheel activity (spontaneous locomotive activity) was significantly reduced in FOXO1 mice compared with control mice. Moreover, the FOXO1 mice showed impaired glycemic control after oral glucose and intraperitoneal insulin administration. These results suggest that FOXO1 negatively regulates skeletal muscle mass and type I fiber gene expression and leads to impaired skeletal muscle function. Activation of FOXO1 may be involved in the pathogenesis of sarcopenia, the age-related decline in muscle mass in humans, which leads to obesity and diabetes. Skeletal muscle is the largest organ in the human body, comprising about 40% of the body weight. The mass and composition of skeletal muscle are critical for its functions, such as exercise, energy expenditure, and glucose metabolism (1Zurlo F. Larson K. Bogardus C. Ravussin E. J. Clin. Investig. 1990; 86: 1423-1427Crossref PubMed Scopus (648) Google Scholar, 2Berchtold M.W. Brinkmeier H. Muntener M. Physiol. Rev. 2000; 80: 1215-1265Crossref PubMed Scopus (697) Google Scholar). Elderly humans are known to undergo a progressive loss of muscle fibers associated with diabetes, obesity, and decreased physical activity (sarcopenia) (3Proctor D. Balagopal P. Nair K. J. Nutr. 1998; 128: S351-S355Crossref PubMed Google Scholar). In human skeletal muscle, there are two major classifications of fiber type: type I (slow-twitch oxidative, so-called red muscle) and type II (fast-twitch glycolytic, so-called white muscle) fibers (2Berchtold M.W. Brinkmeier H. Muntener M. Physiol. Rev. 2000; 80: 1215-1265Crossref PubMed Scopus (697) Google Scholar). Mass, fiber size, and fiber composition in adult skeletal muscle are regulated in response to changes in physical activity, environment, or pathological conditions. For example, space flight experiments using rats showed a reduction in total skeletal muscle mass of up to 37% as well as a significant loss of contractile proteins in type I but not type II fibers by 1-2 weeks of microgravity (4Fitts R. Riley D. Widrick J. J. Exp. Biol. 2001; 204: 3201-3208PubMed Google Scholar). Furthermore, the ratio of type I to type II fibers is associated with obesity and diabetes; the number of type I fibers is reduced in obese subjects and diabetic subjects compared with that in controls (5Hickey M.S. Carey J.O. Azevedo J.L. Houmard J.A. Pories W.J. Israel R.G. Dohm G.L. Am. J. Physiol. 1995; 268: E453-E457PubMed Google Scholar, 6Gaster M. Staehr P. Beck-Nielsen H. Schroder H.D. Handberg A. Diabetes. 2001; 50: 1324-1329Crossref PubMed Scopus (215) Google Scholar, 7Tanner C.J. Barakat H.A. Dohm G.L. 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Systemic administration of IGF-1 results in increased skeletal muscle protein and reduced protein degradation (10Zdanowicz M. Moyse J. Wingertzahn M. O'Connor M. Teichberg S. Slonim A. Endocrinology. 1995; 136: 4880-4886Crossref PubMed Scopus (50) Google Scholar). In addition, overexpression of IGF-1 blocks the age-related loss of skeletal muscle (11Barton-Davis E.R. Shoturma D.I. Musaro A. Rosenthal N. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15603-15607Crossref PubMed Scopus (603) Google Scholar). Supplementation of IGF-1 to muscle cells in vitro promotes myotube hypertrophy, suggesting that hypertrophy can be mediated by autocrine- or paracrine-produced IGF-1 (12Florini J. Ewton D. Coolican S. Endocr. Rev. 1996; 17: 481-517PubMed Google Scholar). Thus, delivery of the IGF-1 gene specifically into skeletal muscle has been proposed as a genetic therapy for skeletal muscle disorders. A better understanding of the role of IGF-1 in skeletal muscle is therefore of great importance. Specialized/differentiated myofiber phenotypes, including type I and type II fibers, are plastic and are physiologically controlled by variations in motor neuron activity. The influence of motor neuron activity on different types of skeletal muscle fibers is considered to be transduced via calcium signaling and downstream molecules such as calcineurin and the calmodulin-dependent kinase (CaMK) pathway (13Stull J.T. J. Biol. Chem. 2001; 276: 2311-2312Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Signals generated by calcium/calcineurin/CaMK augment the transactivating function of Mef2 and/or NFAT and enhance type I fiber-specific gene expression (13Stull J.T. J. Biol. Chem. 2001; 276: 2311-2312Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 14Chin E.R. Olson E.N. Richardson J.A. Yang Q. Humphries C. Shelton J.M. Wu H. Zhu W. Bassel-Duby R. 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We also reproduced the PGC-1α-induced red appearance of skeletal muscle; both type I and type II fibers appear redder in transgenic mice overexpressing PGC-1α in skeletal muscle (23Miura S. Kai Y. Ono M. Ezaki O. J. Biol. Chem. 2003; 278: 31385-31390Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). FOXO1 (FKHR), FOXO4 (AFX), and FOXO3a (FKHRL1) are a subfamily of the forkhead type transcription factors (24Anderson M.J. Viars C.S. Czekay S. Cavenee W.K. Arden K.C. Genomics. 1998; 47: 187-199Crossref PubMed Scopus (287) Google Scholar, 25Kaestner K.H. Knochel W. Martinez D.E. Genes Dev. 2000; 14: 142-146PubMed Google Scholar). FOXO1 was originally cloned from a rhabdomyosarcoma because of its aberrant fusion with another transcription factor, PAX3, resulting from a chromosomal translocation (26Galili N. Davis R.J. Fredericks W.J. Mukhopadhyay S. Rauscher 3rd, F.J. Emanuel B.S. Rovera G. Barr F.G. Nat. Genet. 1993; 5: 230-235Crossref PubMed Scopus (774) Google Scholar). Recent studies have shown that the FOXO protein can also act as a cofactor of nuclear receptor activity (27Schuur E.R. Loktev A.V. Sharma M. Sun Z. Roth R.A. Weigel R.J. J. Biol. Chem. 2001; 276: 33554-33560Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 28Zhao H.H. Herrera R.E. Coronado-Heinsohn E. Yang M.C. Ludes-Meyers J.H. Seybold-Tilson K.J. Nawaz Z. Yee D. Barr F.G. Diab S.G. Brown P.H. Fuqua S.A.W. Osborne C.K. J. Biol. Chem. 2001; 276: 27907-27912Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 29Dowell P. Otto T.C. Adi S. Lane M.D. J. Biol. Chem. 2003; 278: 45485-45491Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 30Hirota K. Daitoku H. Matsuzaki H. Araya N. Yamagata K. Asada S. Sugaya T. Fukamizu A. J. Biol. Chem. 2003; 278: 13056-13060Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). FOXO family members have been shown to regulate various cellular functions. FOXOs influence the transcription of genes involved in metabolism (31Ayala J.E. Streeper R.S. Desgrosellier J.S. Durham S.K. Suwanichkul A. Svitek C.A. Goldman J.K. Barr F.G. Powell D.R. O'Brien R.M. Diabetes. 1999; 48: 1885-1889Crossref PubMed Scopus (100) Google Scholar, 32Barthel A. Schmoll D. Kruger K.D. Bahrenberg G. Walther R. Roth R.A. Joost H.G. Biochem. Biophys. Res. Commun. 2001; 285: 897-902Crossref PubMed Scopus (95) Google Scholar, 33Nakae J. Kitamura T. Silver D.L. Accili D. J. Clin. Investig. 2001; 108: 1359-1367Crossref PubMed Scopus (506) Google Scholar, 34Nadal A. Marrero P.F. Haro D. Biochem. J. 2002; 366: 289-297Crossref PubMed Google Scholar), the cell cycle (35Dijkers P.F. Medema R.H. Pals C. Banerji L. Thomas N.S.B. Lam E.W.F. Burgering B.M.T. Raaijmakers J.A.M. Lammers J.W.J. Koenderman L. Coffer P.J. Mol. Cell. Biol. 2000; 20: 9138-9148Crossref PubMed Scopus (542) Google Scholar, 36Medema R.H. Kops G.J.P.L. Bos J.L. Burgering B.M.T. Nature. 2000; 404: 782-787Crossref PubMed Scopus (1226) Google Scholar), and apoptosis (37Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5434) Google Scholar, 38Kops G.J.P.L. Dansen T.B. Polderman P.E. Saarloos I. Wirtz K.W.A. Coffer P.J. Huang T.T. Bos J.L. Medema R.H. Burgering B.M.T. Nature. 2002; 419: 316-321Crossref PubMed Scopus (1273) Google Scholar). In addition, FOXO1 can modulate cell differentiation; the constitutive active form of FOXO1 prevents the differentiation of preadipocytes (39Nakae J. Kitamura T. Kitamura Y. Biggs III, W.H. Arden K.C. Accili D. Dev. Cell. 2003; 4: 119-129Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar) and stimulates myotube fusion of primary mouse myoblasts (40Bois P.R.J. Grosveld G.C. EMBO J. 2003; 22: 1147-1157Crossref PubMed Scopus (141) Google Scholar). Moreover, a FOXO1 knockout mouse has been reported; Foxo1 haploinsufficiency restores insulin sensitivity and rescues the diabetic phenotype in insulin-resistant mice by reducing the hepatic expression of glucogenetic genes and by increasing the adipocytic expression of insulin-sensitizing genes (41Nakae J. Biggs III, W.H. Kitamura T. Cavenee W.K. Wright C.V. Arden K.C. Accili D. Nat. Genet. 2002; 32: 245-253Crossref PubMed Scopus (532) Google Scholar). We have shown that FOXO1 expression is increased in skeletal muscle in energy-deprived states, such as in fasting mice, in mice with streptozotocin (STZ)-induced diabetes, and in mice after treadmill running (42Kamei Y. Mizukami J. Miura S. Suzuki M. Takahashi N. Kawada T. Taniguchi T. Ezaki O. FEBS Lett. 2003; 536: 232-236Crossref PubMed Scopus (103) Google Scholar). However, the physiological role of FOXO1 in skeletal muscle is still unclear. Although many studies have been performed using cultured cells, studies using animals with genetic modifications focused to the skeletal muscle remain to be conducted in order to understand the function of the FOXO family proteins in vivo. Meanwhile, it has been reported that FOXO1 and PGC-1α can physically interact and regulate gene expression in the liver (43Puigserver P. Rhee J. Donovan J. Walkey C.J. Yoon J.C. Oriente F. Kitamura Y. Altomonte J. Dong H. Accili D. Spiegelman B.M. Nature. 2003; 423: 550-555Crossref PubMed Scopus (1184) Google Scholar). Given that PGC-1α is important for the differentiation of type I fibers, FOXO1 might be involved in this process. (Here-after, we use “differentiation of muscle fiber” to mean “a switch from one fiber type to another fiber type.”) On the other hand, a genetic study of Caenorhabditis elegans showed that DAF16, the worm counterpart of FOXO, functions as a suppressor of insulin receptor-like signaling (44Ogg S. Paradis S. Gottlieb S. Patterson G.I. Lee L. Tissenbaum H.A. Ruvkun G. Nature. 1997; 389: 994-999Crossref PubMed Scopus (1543) Google Scholar). Thus, the FOXO family may act negatively in mammals as a downstream player in insulin or IGF signaling. As IGF-1 plays an important role in controlling skeletal muscle mass, FOXO1 might also be involved in this process. To gain insight into the potential role of FOXO1 in skeletal muscle, including the control of skeletal muscle mass and the control of differentiation of muscle fiber type, we established transgenic mice specifically overexpressing FOXO1 in their skeletal muscle. Most interestingly, these mice showed reduced skeletal muscle mass, and the muscle was paler in color. Histochemical, physiological, and microarray analyses of these FOXO1 transgenic mice showed that FOXO1 is involved in the regulation of skeletal muscle mass and type I fiber gene expression. In addition, our results suggest that FOXO1 activation may play a role in the impairment of skeletal muscle function including glycemic control. RNA Analysis—Northern blot analyses were performed as described previously (42Kamei Y. Mizukami J. Miura S. Suzuki M. Takahashi N. Kawada T. Taniguchi T. Ezaki O. FEBS Lett. 2003; 536: 232-236Crossref PubMed Scopus (103) Google Scholar). The cDNA probes for Gadd45α (GenBank™ accession number, U00937), troponin C (slow) (M29793), troponin T (slow) (AV213431), myosin light chain (MLC) (slow) (M91602), myoglobin (X04405), mitochondrial creatine kinase (mtCK, AV250974), F0,F1-ATPase (AF030559), MLC (fast) (U77943), troponin I (fast) (J04992), troponin T (fast) (L48989), cathepsin L (X06086), IGF-binding protein 5 (IGFBP5) (L12447), MuRF1 (AF294790), and atrogin 1 (AF441120) were obtained by reverse transcription-PCR. The PCR primers used are as follows: Gadd45α, forward, 5′-TCGCACTTGCAATATGACTT-3′, and reverse, 5′-CGGATGCCATCACCGTTCCG-3′; troponin C (slow), forward, 5′-AGCTGCGGTAGAACAGTTGA-3′, and reverse, 5′-TCACCTGTGGCCTGCAGCAT-3′; troponin T (slow), forward, 5′-TTCTGTCCAACATGGGAGCT-3′, and reverse, 5′-TCGGAATTTCTGGGCGTGGC-3′; MLC (slow), forward, 5′-GAGTTCAAGGAAGCCTTCAC-3′, and reverse, 5′-CTGCGAACATCTGGTCGATC-3′; myoglobin, forward, 5′-CACCATGGGGCTCAGTGATG-3′, and reverse, 5′-CTCAGCCCTGGAAGCCTAGC-3′; mtCK, forward, 5′-AAAGGAAGTGGAACGATTAA-3′, and reverse, 5′-TTGATGTCTTGGCCTCTCTC-3′,F0,F1-ATPase, forward, 5′-ACTGACCCTGCCCCTGCAAC-3′, and reverse, 5′-CAAGGCTCTTGTGTGGCCTG-3′, MLC (fast), forward, 5′-AGGGATGGCATTATCGACAA-3′, and reverse, 5′-CAGATGTTCTTGTAGTCCAC-3′; troponin I, (fast), forward, 5′-AGGAAAGCCGCCGAGAATCT-3′, and reverse, 5′-TACTGGGGAAGTGGGCAGTT-3′; troponin T (fast), forward, 5′-CAGCAAAGAATTCGCGCTGA-3′, and reverse, 5′-GGCCTTCTTGCTGTGCTTCT-3′; cathepsin L, forward, 5′-CGGAGGAGTCTTACCCCTAT-3′, and reverse, 5′-CTACCCATCAATTCACGACA-3′; IGFBP5, forward, 5′-GCCTATGCCGTACCGGCTCA-3′, and reverse, 5′-CTTCACAGCCTCAGCCTTCA-3′; MuRF1, forward, 5′-ATGAACTTCACGGTGGGTTT-3′, and reverse, 5′-TCAGTGCAGGCCTGAGCCTT-3′; and atrogin 1, forward, 5′-ATGCCGTTCCTTGGGCAGGA-3′, and reverse, 5′-TCAGAACTTGAACAAATTGA-3′. FOXO1, FOXO3a, and FOXO4 cDNA probes were prepared as reported previously (42Kamei Y. Mizukami J. Miura S. Suzuki M. Takahashi N. Kawada T. Taniguchi T. Ezaki O. FEBS Lett. 2003; 536: 232-236Crossref PubMed Scopus (103) Google Scholar). COXII, COXIV, Mef2c, PGC-1α, and glucose transporter 4 cDNA probes were prepared as described previously (23Miura S. Kai Y. Ono M. Ezaki O. J. Biol. Chem. 2003; 278: 31385-31390Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). NFAT (IMAGE clone 4109469) and CaMK IIβ (IMAGE clone 5014712) cDNA probes were purchased from Invitrogen. Generating Transgenic Mice—The human skeletal muscle α-actin promoter (45Brennan K.J. Hardeman E.C. J. Biol. Chem. 1993; 268: 719-725Abstract Full Text PDF PubMed Google Scholar) was provided by Drs. E. D. Hardeman and K. Guven (Children's Medical Research Institute, Australia). The human FOXO1 cDNA was as described previously (42Kamei Y. Mizukami J. Miura S. Suzuki M. Takahashi N. Kawada T. Taniguchi T. Ezaki O. FEBS Lett. 2003; 536: 232-236Crossref PubMed Scopus (103) Google Scholar). The transgene (Fig. 1A) was excised from agarose gel and purified for injection (2 ng μl-1). Fertilized eggs were recovered from C57BL/6 females crossed with C57BL/6 males and microinjected at Japan SLC Inc. (Hamamatsu, Japan). The mice were maintained at a constant temperature of 22 °C with fixed artificial light (12-h light and 12-h dark cycle). Care of the mice was conducted in accordance with the institutional guidelines. Body Composition Analysis—Mice were anesthetized with pentobarbital sodium, Nembutal (0.08 mg/g body weight, Abbott), and scanned with a Lunar PIXI mus2 densitometer (Lunar Corp., Madison, WI), equipped for dual energy x-ray absorptiometry (DEXA) (46Nagy T.R. Clair A.L. Obes. Res. 2000; 8: 392-398Crossref PubMed Scopus (245) Google Scholar). Immunoblotting—Protein extracts from skeletal muscle were prepared by centrifugation of the tissue homogenates as described previously (47Hahn C.G. Covault J. Anal. Biochem. 1990; 190: 193-197Crossref PubMed Scopus (27) Google Scholar). Protein extracts (30 μg) separated by SDS-PAGE were electrophoretically transferred to Immobilon P membranes (Millipore, Bedford, MA). Immunoblotting was performed by using goat anti-FOXO1 IgG (N-18, Santa Cruz Biotechnology, Inc. Santa Cruz, CA), goat anti-troponin I (slow) (C-19, Santa Cruz Biotechnology), goat anti-troponin I (fast) (C-19, Santa Cruz Biotechnology), goat anti-myoglobin (M-109, Santa Cruz Biotechnology), or rabbit anti-PGC-1α (C terminus, Calbiochem) as primary antibodies (1:1000) and anti-goat IgG or anti-rabbit IgG conjugated with horseradish peroxidase as secondary antibodies (1:1000). Bands were visualized with the enhanced chemiluminescence system (Amersham Biosciences). Histological Analyses—Skeletal muscle (soleus) samples were frozen in liquid nitrogen-cooled isopentane, and transverse serial sections were stained with ATPase at pH 4.3 to detect type I fibers and at pH 10.5 to detect type II fibers (48Ogilvie R.W. Feeback D.L. Stain Technol. 1990; 65: 231-241Crossref PubMed Scopus (93) Google Scholar). The ratio of type I fibers to type II fibers and the size (area) of skeletal muscle cells were determined by counting cell numbers in six randomly selected cross-section areas (each 900 μm2) stained with ATPase at pH 4.3. Blood Analysis—Blood samples were obtained from mice tail tips for hormone and metabolite determination under feeding conditions. Immunoreactive insulin was measured by an insulin assay kit (Morinaga, Kanagawa, Japan), free fatty acid by NEFA C-test Wako (Wako Biochemicals, Osaka, Japan), lactate by the lactate reagent (Sigma), and glucose by the TIDEX glucose analyzer (Sankyo, Tokyo, Japan). Running Wheel Activity—Mice were housed individually in cages (9 × 22 × 9 cm) equipped with a running wheel (20-cm in diameter, Shinano Co., Tokyo, Japan). Each wheel revolution was registered by a magnetic switch, which was connected to a counter. The number of revolutions was recorded daily for 6 days. Oral Glucose and Insulin Tolerance Test—For the oral glucose tolerance test, d-glucose (1 mg/g of body weight, 10% (w/v) glucose solution) was administered with a stomach tube after an overnight fast. Blood samples were obtained by cutting the tail tip before and 30, 60, and 120 min after glucose administration. For the insulin tolerance test, human insulin (Humulin R; Lilly) was injected intraperitoneally (0.75 milliunits/g of body weight) into fed animals. Blood glucose concentrations were measured using a TIDEX glucose analyzer (Sankyo, Tokyo, Japan). Microarray Analyses—RNA was isolated from skeletal muscle (quadriceps) of sex- and age-matched FOXO1 mice (A1 and A2 lines) and control mice (males at 4 months of age, RNA from three mice of each group were combined). Each of the combined samples was hybridized to the Affymetrix MGU74A microarray, which contains 12,489 genes including ESTs, and analyzed with the Affymetrix Gene Chip 3.1 software as described previously (49Takahashi M. Tsuboyama-Kasaoka N. Nakatani T. Ishii M. Tsutsumi S. Aburatani H. Ezaki O. Am. J. Physiol. 2002; 282: G338-G348Crossref PubMed Scopus (18) Google Scholar). Of the 12,489 genes including ESTs analyzed, 2500 (nontransgenic control mice), 2490 (line A1, transgenic), and 2510 (line A2, transgenic) genes were expressed at a substantial level (absolute call is present and average difference is above 150). Genes were classified on the basis of the biological function of the encoded protein, using a previously established classification scheme (50Adams M.D. Kerlavage A.R. Fleischmann R.D. Fuldner R.A. Bult C.J. Lee N.H. Kirkness E.F. Weinstock K.G. Gocayne J.D. White O. et al.Nature. 1995; 377: 3-174PubMed Google Scholar). The classification scheme was composed of seven major functional categories and several minor functional categories within the major categories. Statistical Analyses—Statistical comparisons of data from the experimental groups were performed by the one-way analysis of variance, and groups were compared using the Fisher's protected least significant difference test (Statview 5.0, Abacus Concepts, Inc., Berkeley, CA). The glucose and insulin tolerance curves were compared by repeated measure analysis (Statview 5.0, Abacus Concepts). When significant, groups were compared by the Fisher's protected least significant difference test. Statistical significance was defined as p < 0.05. Creation of FOXO1 Mice—The human skeletal muscle α-actin promoter (45Brennan K.J. Hardeman E.C. J. Biol. Chem. 1993; 268: 719-725Abstract Full Text PDF PubMed Google Scholar) was used to drive the expression of the human FOXO1 transgene in mice (Fig. 1A). During development, cardiac muscle α-actin is the predominant isoform of sarcomeric α-actin in mice, and the switch to skeletal muscle α-actin occurs postpartum (45Brennan K.J. Hardeman E.C. J. Biol. Chem. 1993; 268: 719-725Abstract Full Text PDF PubMed Google Scholar). Thus, by using the skeletal muscle α-actin promoter, the possibility that embryonic expression of FOXO1 might interfere with development was minimized. We obtained two independent lines of transgenic mice (lines A1 and A2). Southern blot analysis of DNA obtained from mouse tails was performed as shown in Fig. 1B. The transgene copy number of each animal was estimated by densitometric scanning of the autoradiographs from the Southern blots. Expression of the FOXO1 transgene was evaluated by Northern blot analysis with RNA isolated from the tissues of FOXO1 mice and age-matched control mice at 8 weeks of age (Fig. 1C). The use of this promoter resulted in predominantly high expression levels of the FOXO1 transgene in skeletal muscle (about 3.5 kb). The A2 line showed expression levels of the FOXO1 transgene in skeletal muscle that were similar to or slightly higher than that in the A1 line. Transgene expression was observed not only in the gastrocnemius and quadriceps but also in other areas of skeletal muscle including the tibialis anterior, extensor digitorum longus (EDL), and soleus (not shown). The blot was then re-hybridized with a cDNA probe of Gadd45α, an authentic target gene of FOXO1 (51Furukawa-Hibi Y. Yoshida-Araki K. Ohta T. Ikeda K. Motoyama N. J. Biol. Chem. 2002; 277: 26729-26732Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 52Tran H. Brunet A. Grenier J.M. Datta S.R. Fornace Jr., A.J. DiStefano P.S. Chiang L.W. Greenberg M.E. Science. 2002; 296: 530-534Crossref PubMed Scopus (710) Google Scholar). As expected, induction of the expression of Gadd45α was observed in skeletal muscle but not in other tissues in both FOXO1 transgenic mouse lines (Fig. 1C), indicating that the transgene expressed a functional FOXO1 protein. By using an antibody that recognizes both human and mouse FOXO1, we confirmed the presence of the FOXO1 protein in the skeletal muscle of FOXO1 mice (Fig. 1D). An ∼2.2-fold (line A1) and 3-fold (line A2) increase in FOXO1 protein levels was observed. These increases were at the physiological level, since 24-h fasting has been shown to increase FOXO1 protein content by 2.5-3-fold (Ref. 53Furuyama T. Kitayama K. Yamashita H. Mori N. Biochem. J. 2003; 375: 365-371Crossref PubMed Scopus (260) Google Scholar and data not shown). FOXO1 Mice Are Small—The apparent phenotype observed in FOXO1 mice was small stature and thinner legs than the control mice. Both male and female transgenic mice weighed about 10% less than the control mice at 5 weeks of age (not shown). We used DEXA to measure the lean body mass (body weight excluding fat weight) and the content of fat in the whole body of the A1 line (at 5 months of age) and the A2 line (at 4 months of age) in age- and sex-matched control mice (Table I). Both body weight and lean body mass were significantly lower in both male and female FOXO1 mice (both lines) than in control mice. However, the fat content per total body weight of both FOXO1 mouse lines was comparable with that of nontransgenic mice (Table I). Thus, the decrease in body weight of the FOXO1 mice is not caused by a decrease in body fat but by a decrease in lean body mass. Consistent with the data on decreased lean body mass, the skeletal muscles in FOXO1 mice were smaller in size and dry mass, as well as paler in color than those of control mice (Fig. 1E). Consumption of food per body weight was not significantly different between FOXO1 mice and control mice (Table I). Blood metabolite (free fatty acid, lactate, and glucose) and insulin levels did not differ significantly between FOXO1 mice and the controls (Table I).Table IFOXO1 mice are smaller in body weight and lean body massMiceNumbersSexAgeBody weightLean body massFat contentFood intakeFree fatty acidLactateGlucoseInsulingg%g/g/daymEq/litermg/mlmg/dlpg/mlControl4Male5 months29.0 ± 1.024.1 ± 0.320.8 ± 1.60.18 ± 0.0050.30 ± 0.02553.0 ± 4.3163 ± 2.91775 ± 700A14Male24.5 ± 0.4ap < 0.01.20.3 ± 0.4bp < 0.001.20.8 ± 0.50.17 ± 0.0040.34 ± 0.09856.3 ± 8.3173 ± 14739 ± 139Control4Female21.6 ± 0.919.3 ± 0.912.9 ± 1.00.25 ± 0.0170.39 ± 0.06032.7 ± 3.1158 ± 8.0289 ± 14A16Female18.4 ± 0.4ap < 0.01.16.4 ± 0.2ap < 0.01.15.8 ± 1.20.24 ± 0.0170.38

The primary muscle disorders are a diverse group of diseases caused by various defective structural proteins, abnormal signaling molecules, enzymes and proteins involved in posttranslational modifications, and other mechanisms. Although there is increasing clarification of the primary aberrant cellular processes responsible for these conditions, the decisive factors involved in the secondary pathogenic cascades are still mainly obscure. Given the emerging roles of microRNAs (miRNAs) in modulation of cellular phenotypes, we searched for miRNAs regulated during the degenerative process of muscle to gain insight into the specific regulation of genes that are disrupted in pathological muscle conditions. We describe 185 miRNAs that are up- or down-regulated in 10 major muscular disorders in humans [Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophies types 2A and 2B, Miyoshi myopathy, nemaline myopathy, polymyositis, dermatomyositis, and inclusion body myositis]. Although five miRNAs were found to be consistently regulated in almost all samples analyzed, pointing to possible involvement of a common regulatory mechanism, others were dysregulated only in one disease and not at all in the other disorders. Functional correlation between the predicted targets of these miRNAs and mRNA expression demonstrated tight posttranscriptional regulation at the mRNA level in DMD and Miyoshi myopathy. Together with direct mRNA–miRNA predicted interactions demonstrated in DMD, some of which are involved in known secondary response functions and others that are involved in muscle regeneration, these findings suggest an important role of miRNAs in specific physiological pathways underlying the disease pathology.

Caveolae are invaginations of the plasma membrane involved in many cellular processes, including clathrin-independent endocytosis, cholesterol transport, and signal transduction. They are characterized by the presence of caveolin proteins. Mutations that cause deficiency in caveolin-3, which is expressed exclusively in skeletal and cardiac muscle, have been linked to muscular dystrophy. Polymerase I and transcript release factor (PTRF; also known as cavin) is a caveolar-associated protein suggested to play an essential role in the formation of caveolae and the stabilization of caveolins. Here, we identified PTRF mutations in 5 nonconsanguineous patients who presented with both generalized lipodystrophy and muscular dystrophy. Muscle hypertrophy, muscle mounding, mild metabolic complications, and elevated serum creatine kinase levels were observed in these patients. Skeletal muscle biopsies revealed chronic dystrophic changes, deficiency and mislocalization of all 3 caveolin family members, and reduction of caveolae structure. We generated expression constructs recapitulating the human mutations; upon overexpression in myoblasts, these mutations resulted in PTRF mislocalization and disrupted physical interaction with caveolins. Our data confirm that PTRF is essential for formation of caveolae and proper localization of caveolins in human cells and suggest that clinical features observed in the patients with PTRF mutations are associated with a secondary deficiency of caveolins.

•Three distinct subtypes of immune-mediated necrotizing myopathies are defined.•New pathological criteria for immune-mediated necrotizing myopathies are defined.•Anti-HMGCR myopathy, anti-SRP myopathy and antibody negative IMNM are defined.•Therapeutic recommendations for anti-HMGCR myopathy, anti-SRP myopathy are given.

Fatty and fibrous connective tissue formation is a hallmark of diseased skeletal muscle and deteriorates muscle function. We previously identified non-myogenic mesenchymal progenitors that contribute to adipogenesis and fibrogenesis in mouse skeletal muscle. In this study, we report the identification and characterization of a human counterpart to these progenitors. By using PDGFRα as a specific marker, mesenchymal progenitors can be identified in the interstitium and isolated from human skeletal muscle. PDGFRα(+) cells represent a cell population distinct from CD56(+) myogenic cells, and adipogenic and fibrogenic potentials were highly enriched in the PDGFRα(+) population. Activation of PDGFRα stimulates proliferation of PDGFRα(+) cells through PI3K-Akt and MEK2-MAPK signaling pathways, and aberrant accumulation of PDGFRα(+) cells was conspicuous in muscles of patients with both genetic and non-genetic muscle diseases. Our results revealed the pathological relevance of PDGFRα(+) mesenchymal progenitors to human muscle diseases and provide a basis for developing therapeutic strategy to treat muscle diseases.

<h3>Objective</h3> To elucidate the common and distinct clinical features of immune-mediated necrotising myopathy (IMNM), also known as necrotising autoimmune myopathy associated with autoantibodies against signal recognition particle (SRP) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR). <h3>Methods</h3> We examined a cohort of 460 patients with idiopathic inflammatory myopathies (IIMs) through a muscle biopsy-oriented registration study in Japan. Study entry was strictly determined by the comprehensive histological assessment to exclude other neuromuscular disorders. Anti-SRP and anti-HMGCR antibodies were detected by RNA immunoprecipitation and ELISA, respectively. <h3>Results</h3> Of 460 patients with IIM, we diagnosed 73 (16%) as having inclusion body myositis (IBM). Of 387 patients with IIMs other than IBM, the frequencies of anti-SRP and anti-HMGCR antibodies were 18% and 12%, respectively. One patient had both autoantibodies. Severe limb muscle weakness, neck weakness, dysphagia, respiratory insufficiency and muscle atrophy were more frequently observed in patients with anti-SRP antibodies than in those with anti-HMGCR antibodies. Serum creatine levels were markedly higher in the patients with autoantibodies than in those without. Histology was characterised by necrosis and regeneration of muscle fibres and was consistent with IMNM except in 1 HMGCR-positive IBM patient. Most patients were initially treated with corticosteroids; however, additional immunosuppressive drugs were required, especially in the patients with anti-SRP antibodies. Rates of unsatisfactory neurological outcome were similar in the 2 autoantibody groups. <h3>Conclusions</h3> Anti-SRP antibodies are associated with severe neurological symptoms, more so than are anti-HMGCR antibodies. Although these autoantibodies are independent serological markers associated with IMNM, patients bearing either share common characteristics.

Facioscapulohumeral dystrophy (FSHD) is associated with somatic chromatin relaxation of the D4Z4 repeat array and derepression of the D4Z4-encoded <i>DUX4</i> retrogene coding for a germline transcription factor. Somatic <i>DUX4</i> derepression is caused either by a 1–10 unit repeat-array contraction (FSHD1) or by mutations in <i>SMCHD1</i>, which encodes a chromatin repressor that binds to D4Z4 (FSHD2). Here, we show that heterozygous mutations in DNA methyltransferase 3B (<i>DNMT3B</i>) are a likely cause of D4Z4 derepression associated with low levels of <i>DUX4</i> expression from the D4Z4 repeat and increased penetrance of FSHD. Recessive mutations in <i>DNMT3B</i> were previously shown to cause immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome. This study suggests that transcription of <i>DUX4</i> in somatic cells is modified by variations in its epigenetic state and provides a basis for understanding the reduced penetrance of FSHD within families.

Nemaline myopathy (NEM) is a common congenital myopathy. At the very severe end of the NEM clinical spectrum are genetically unresolved cases of autosomal-recessive fetal akinesia sequence. We studied a multinational cohort of 143 severe-NEM-affected families lacking genetic diagnosis. We performed whole-exome sequencing of six families and targeted gene sequencing of additional families. We identified 19 mutations in KLHL40 (kelch-like family member 40) in 28 apparently unrelated NEM kindreds of various ethnicities. Accounting for up to 28% of the tested individuals in the Japanese cohort, KLHL40 mutations were found to be the most common cause of this severe form of NEM. Clinical features of affected individuals were severe and distinctive and included fetal akinesia or hypokinesia and contractures, fractures, respiratory failure, and swallowing difficulties at birth. Molecular modeling suggested that the missense substitutions would destabilize the protein. Protein studies showed that KLHL40 is a striated-muscle-specific protein that is absent in KLHL40-associated NEM skeletal muscle. In zebrafish, klhl40a and klhl40b expression is largely confined to the myotome and skeletal muscle, and knockdown of these isoforms results in disruption of muscle structure and loss of movement. We identified KLHL40 mutations as a frequent cause of severe autosomal-recessive NEM and showed that it plays a key role in muscle development and function. Screening of KLHL40 should be a priority in individuals who are affected by autosomal-recessive NEM and who present with prenatal symptoms and/or contractures and in all Japanese individuals with severe NEM.

Nemaline myopathy (NM) is a genetic muscle disorder characterized by muscle dysfunction and electron-dense protein accumulations (nemaline bodies) in myofibers. Pathogenic mutations have been described in 9 genes to date, but the genetic basis remains unknown in many cases. Here, using an approach that combined whole-exome sequencing (WES) and Sanger sequencing, we identified homozygous or compound heterozygous variants in LMOD3 in 21 patients from 14 families with severe, usually lethal, NM. LMOD3 encodes leiomodin-3 (LMOD3), a 65-kDa protein expressed in skeletal and cardiac muscle. LMOD3 was expressed from early stages of muscle differentiation; localized to actin thin filaments, with enrichment near the pointed ends; and had strong actin filament-nucleating activity. Loss of LMOD3 in patient muscle resulted in shortening and disorganization of thin filaments. Knockdown of lmod3 in zebrafish replicated NM-associated functional and pathological phenotypes. Together, these findings indicate that mutations in the gene encoding LMOD3 underlie congenital myopathy and demonstrate that LMOD3 is essential for the organization of sarcomeric thin filaments in skeletal muscle.

Anti-signal recognition particle (SRP) antibodies are used as serological markers of necrotizing myopathy, which is characterized by many necrotic and regenerative muscle fibers without or with minimal inflammatory cell infiltration. The clinical spectrum associated with anti-SRP antibodies seems to be broad. To describe the clinical characteristics, autoantibodies status, and neurological outcome associated with anti-SRP antibody. We studied clinical and laboratory findings of 100 patients with inflammatory myopathy and anti-SRP antibodies. Anti-SRP antibodies in serum were detected by the presence of 7S RNA using RNA immunoprecipitation. In addition, enzyme-linked immunosorbent assays (ELISAs) using a 54-kD protein of SRP (SRP54) and 3-hydroxyl-3-methylglutatyl-coenzyme A reductase (HMGCR) were also conducted. The mean onset age of the 61 female and 39 male patients was 51 years (range 4–82 years); duration ≥ 12 months before diagnosis was seen in 23 cases. All patients presented limbs weakness; 63 had severe weakness, 70 neck weakness, 41 dysphagia, and 66 muscle atrophy. Extramuscular symptoms and associated disorders were infrequent. Creatine kinase levels were mostly more than 1000 IU/L. Histological diagnosis showed 84 patients had necrotizing myopathy, and apparent cell infiltration was observed in 16 patients. Anti-SRP54 antibodies were undetectable in 18 serum samples with autoantibodies to 7S RNA. Anti-HMGCR antibodies were positive in 3 patients without the statin treatment, however, were negative in 5 patients with statin-exposure at disease onset. All but 3 patients were treated by corticosteroids and 62 (77 %) of these 81 patients required additional immunotherapy. After 2-years treatment, 22 (27 %) of these 81 patients had poor neurological outcomes with modified Rankin scale scores of 3–5. Multivariate analysis revealed that pediatric disease onset was associated with the poor outcomes. Anti-SRP antibodies are associated with different clinical courses and histological presentations.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive human disease due to mutations in the thymidine phosphorylase (TP) gene.TP enzyme catalyzes the reversible phosphorolysis of thymidine to thymine and 2-deoxy-D-ribose 1-phosphate.We present evidence that thymidine metabolism is altered in MNGIE.TP activities in buffy coats were reduced drastically in all 27 MNGIE patients compared with 19 controls.All MNGIE patients had much higher plasma levels of thymidine than normal individuals and asymptomatic TP mutation carriers.In two patients, the renal clearance of thymidine was ϳ20% that of creatinine, and because hemodialysis demonstrated that thymidine is ultrafiltratable, most of the filtered thymidine is likely to be reabsorbed by the kidney.In vitro, fibroblasts from controls catabolized thymidine in medium; by contrast, MNGIE fibroblasts released thymidine.In MNGIE, severe impairment of TP enzyme activity leads to increased plasma thymidine.In patients who are suspected of having MNGIE, determination of TP activity in buffy coats and thymidine levels in plasma are diagnostic.We hypothesize that excess thymidine alters mitochondrial nucleoside and nucleotide pools leading to impaired mitochondrial DNA replication, repair, or both.Therapies to reduce thymidine levels may be beneficial to MNGIE patients.

Distal myopathy with rimmed vacuoles (DMRV) is an autosomal-recessive disorder with preferential involvement of the tibialis anterior muscle that starts in young adulthood and spares quadriceps muscles. The disease locus has been mapped to chromosome 9p1-q1, the same region as the hereditary inclusion body myopathy (HIBM) locus. HIBM was originally described as rimmed vacuole myopathy sparing the quadriceps; therefore, the two diseases have been suspected to be allelic. Recently, HIBM was shown to be associated with the mutations in the gene encoding the bifunctional enzyme, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE).To determine whether DMRV and HIBM are allelic.The GNE gene was sequenced in 34 patients with DMRV. The epimerase activity in lymphocytes from eight DMRV patients was also measured.The authors identified 27 unrelated DMRV patients with homozygous or compound-heterozygous mutations in the GNE gene. DMRV patients had markedly decreased epimerase activity.DMRV is allelic to HIBM. Various mutations are associated with DMRV in Japan. The loss-of-function mutations in the GNE gene appear to cause DMRV/HIBM.

Danon disease is due to primary deficiency of lysosome-associated membrane protein-2.To define the clinicopathologic features of Danon disease.The features of 20 affected men and 18 affected women in 13 families with genetically confirmed Danon disease were reviewed.All patients had cardiomyopathy, 18 of 20 male patients (90%) and 6 of 18 female patients (33%) had skeletal myopathy, and 14 of 20 male patients (70%) and one of 18 female patients (6%) had mental retardation. Men were affected before age 20 years whereas most affected women developed cardiomyopathy in adulthood. Muscle histology revealed basophilic vacuoles that contain acid phosphatase-positive material within membranes that lack lysosome-associated membrane protein-2. Heart transplantation is the most effective treatment for the otherwise lethal cardiomyopathy.Danon disease is an X-linked dominant multisystem disorder affecting predominantly cardiac and skeletal muscles.

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.

Perlecan, a large heparan sulfate proteoglycan, is a component of the basement membrane and other extracellular matrices and has been implicated in multiple biological functions. Mutations in the perlecan gene (<i>HSPG2</i>) cause two classes of skeletal disorders: the relatively mild Schwartz-Jampel syndrome (SJS) and severe neonatal lethal dyssegmental dysplasia, Silverman-Handmaker type (DDSH). SJS is an autosomal recessive skeletal dysplasia characterized by varying degrees of myotonia and chondrodysplasia, and patients with SJS survive. The molecular mechanism underlying the chondrodystrophic myotonia phenotype of SJS is unknown. In the present report, we identify five different mutations that resulted in various forms of perlecan in three unrelated patients with SJS. Heterozygous mutations in two patients with SJS either produced truncated perlecan that lacked domain V or significantly reduced levels of wild-type perlecan. The third patient had a homozygous 7-kb deletion that resulted in reduced amounts of nearly full-length perlecan. Unlike DDSH, the SJS mutations result in different forms of perlecan in reduced levels that are secreted to the extracellular matrix and are likely partially functional. These findings suggest that perlecan has an important role in neuromuscular function and cartilage formation, and they define the molecular basis involved in the difference in the phenotypic severity between DDSH and SJS.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a multisystemic autosomal recessive disease due to primary thymidine phosphorylase (TP) deficiency. To restore TP activity, we performed reduced intensity allogeneic stem cell transplantations (alloSCTs) in two patients. In the first, alloSCT failed to engraft, but the second achieved mixed donor chimerism, which partially restored buffy coat TP activity and lowered plasma nucleosides. Thus, alloSCT can correct biochemical abnormalities in the blood of patients with MNGIE, but clinical efficacy remains unproven.

Nemaline myopathy (NM) is a rare congenital muscle disorder primarily affecting skeletal muscles that results in neonatal death in severe cases as a result of associated respiratory insufficiency. NM is thought to be a disease of sarcomeric thin filaments as six of eight known genes whose mutation can cause NM encode components of that structure, however, recent discoveries of mutations in non-thin filament genes has called this model in question. We performed whole-exome sequencing and have identified recessive small deletions and missense changes in the Kelch-like family member 41 gene (KLHL41) in four individuals from unrelated NM families. Sanger sequencing of 116 unrelated individuals with NM identified compound heterozygous changes in KLHL41 in a fifth family. Mutations in KLHL41 showed a clear phenotype-genotype correlation: Frameshift mutations resulted in severe phenotypes with neonatal death, whereas missense changes resulted in impaired motor function with survival into late childhood and/or early adulthood. Functional studies in zebrafish showed that loss of Klhl41 results in highly diminished motor function and myofibrillar disorganization, with nemaline body formation, the pathological hallmark of NM. These studies expand the genetic heterogeneity of NM and implicate a critical role of BTB-Kelch family members in maintenance of sarcomeric integrity in NM.

Abstract Objective The fukutin gene ( FKTN ) is the causative gene for Fukuyama‐type congenital muscular dystrophy, characterized by rather homogeneous clinical features of severe muscle wasting and hypotonia from early infancy with mental retardation. In contrast with the severe dystrophic involvement of skeletal muscle, cardiac insufficiency is quite rare. Fukuyama‐type congenital muscular dystrophy is one of the disorders associated with glycosylation defects of α‐dystroglycan, an indispensable molecule for intra‐extra cell membrane linkage. Methods Protein and functional analyses of α‐dystroglycan and mutation screening of FKTN and other associated genes were performed. Results Surprisingly, we identified six patients in four families showing dilated cardiomyopathy with no or minimal limb girdle muscle involvement and normal intelligence, associated with a compound heterozygous FKTN mutation. One patient died by rapid progressive dilated cardiomyopathy at 12 years old, and the other patient received cardiac implantation at 18 years old. Skeletal muscles from the patients showed minimal dystrophic features but have altered glycosylation of α‐dystroglycan and reduced laminin binding ability. One cardiac muscle that underwent biopsy showed altered glycosylation of α‐dystroglycan similar to that observed in a Fukuyama‐type congenital muscular dystrophy patient. Interpretation FKTN mutations could cause much wider spectrum of clinical features than previously perceived, including familial dilated cardiomyopathy and mildest limb girdle muscular dystrophy. Ann Neurol 2006

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.

The store-operated Ca(2+) release-activated Ca(2+) (CRAC) channel is activated by diminished luminal Ca(2+) levels in the endoplasmic reticulum and sarcoplasmic reticulum (SR), and constitutes one of the major Ca(2+) entry pathways in various tissues. Tubular aggregates (TAs) are abnormal structures in the skeletal muscle, and although their mechanism of formation has not been clarified, altered Ca(2+) homeostasis related to a disordered SR is suggested to be one of the main contributing factors. TA myopathy is a hereditary muscle disorder that is pathologically characterized by the presence of TAs. Recently, dominant mutations in the STIM1 gene, encoding a Ca(2+) sensor that controls CRAC channels, have been identified to cause tubular aggregate myopathy (TAM). Here, we identified heterozygous missense mutations in the ORAI1 gene, encoding the CRAC channel itself, in three families affected by dominantly inherited TAM with hypocalcemia. Skeletal myotubes from an affected individual and HEK293 cells expressing mutated ORAI1 proteins displayed spontaneous extracellular Ca(2+) entry into cells without diminishment of luminal Ca(2+) or the association with STIM1. Our results indicate that STIM1-independent activation of CRAC channels induced by dominant mutations in ORAI1 cause altered Ca(2+) homeostasis, resulting in TAM with hypocalcemia.

GNE myopathy is an autosomal recessive muscle disease caused by biallelic mutations in <i>GNE</i>, a gene encoding for a single protein with key enzymatic activities, UDP-N-acetylglucosamine 2-epimerase and N-acetylmannosamine kinase, in sialic acid biosynthetic pathway. The diagnosis should be considered primarily in patients presenting with distal weakness (foot drop) in early adulthood (other onset symptoms are possible too). The disease slowly progresses to involve other lower and upper extremities’ muscles, with marked sparing of the quadriceps. Characteristic findings on biopsies of affected muscles include ‘rimmed’ (autophagic) vacuoles, aggregation of various proteins and fibre size variation. The diagnosis is confirmed by sequencing of the <i>GNE</i> gene. Note that we use a new mutation nomenclature based on the longest transcript (GenBank: NM_001128227), which encodes a 31-amino acid longer protein than the originally described one (GenBank: NM_005476), which has been used previously in most papers. Based upon the pathophysiology of the disease, recent clinical trials as well as early gene therapy trials have evaluated the use of sialic acid or <i>N</i>-acetylmannosamine (a precursor of sialic acid) in patients with GNE myopathy. Now that therapies are under investigation, it is critical that a timely and accurate diagnosis is made in patients with GNE myopathy.

Replication and repair of DNA require equilibrated pools of deoxynucleoside triphosphate precursors. This concept has been proven by in vitro studies over many years, but in vivo models are required to demonstrate its relevance to multicellular organisms and to human diseases. Accordingly, we have generated thymidine phosphorylase (TP) and uridine phosphorylase (UP) double knockout (TP(-/-)UP(-/-)) mice, which show severe TP deficiency, increased thymidine and deoxyuridine in tissues and elevated mitochondrial deoxythymidine triphosphate. As consequences of the nucleotide pool imbalances, brains of mutant mice developed partial depletion of mtDNA, deficiencies of respiratory chain complexes and encephalopathy. These findings largely account for the pathogenesis of mitochondrial neurogastrointestinal encephalopathy (MNGIE), the first inherited human disorder of nucleoside metabolism associated with somatic DNA instability.

<h2>Abstract</h2> Multiple acyl-CoA dehydrogenase deficiency (MADD) is a metabolic disorder due to dysfunction of electron transfer flavoprotein (ETF) or ETF-ubiquinone oxidoreductase (ETF-QO). Mutations in <i>ETFDH</i>, encoding ETF-QO have been associated with both riboflavin-responsive and non-responsive MADD as well as a myopathic form of CoQ₁₀ deficiency, although pathomechanisms responsible for these different phenotypes are not well-defined. We performed mutation analysis in four Taiwanese MADD patients. Three novel <i>ETFDH</i> mutations were identified in four patients and all harbored the p.A84T mutation. Muscle CoQ₁₀ levels and respiratory chain activities measured in two patients were normal. Three patients improved on riboflavin together with carnitine. Our results show that not all MADD patients have CoQ₁₀ deficiency. Based upon our data, riboflavin and carnitine may be the first-line treatment for MADD.

Congenital muscular dystrophy is a heterogeneous group of inherited muscle diseases characterized clinically by muscle weakness and hypotonia in early infancy. A number of genes harboring causative mutations have been identified, but several cases of congenital muscular dystrophy remain molecularly unresolved. We examined 15 individuals with a congenital muscular dystrophy characterized by early-onset muscle wasting, mental retardation, and peculiar enlarged mitochondria that are prevalent toward the periphery of the fibers but are sparse in the center on muscle biopsy, and we have identified homozygous or compound heterozygous mutations in the gene encoding choline kinase beta (CHKB). This is the first enzymatic step in a biosynthetic pathway for phosphatidylcholine, the most abundant phospholipid in eukaryotes. In muscle of three affected individuals with nonsense mutations, choline kinase activities were undetectable, and phosphatidylcholine levels were decreased. We identified the human disease caused by disruption of a phospholipid de novo biosynthetic pathway, demonstrating the pivotal role of phosphatidylcholine in muscle and brain.

Abstract Objective: Emery‐Dreifuss muscular dystrophy (EDMD) is a genetically heterogeneous muscular disease that presents with muscular dystrophy, joint contractures, and cardiomyopathy with conduction defects. Mutations in several nuclear envelope protein genes have been associated with EDMD in less than half of patients, implying the existence of other causative and modifier genes. We therefore analyzed TMEM43 , which encodes LUMA, a newly identified nuclear membrane protein and also a binding partner of emerin and lamins, to investigate whether LUMA may contribute to the pathomechanism of EDMD‐related myopathy. Methods: Forty‐one patients with EDMD‐related myopathy were enrolled. In vitro and in vivo transfection analyses were performed to assay the binding partners and oligomerization of mutant LUMA. Results: We identified heterozygous missense mutations, p.Glu85Lys and p.Ile91Val in TMEM43 , in 2 EDMD‐related myopathy patients. Reduced nuclear staining of LUMA was observed in the muscle from the patient with p.Glu85Lys mutation. By in vitro transfection analysis, p.Glu85Lys mutant LUMA resulted to failure in oligomerization, a process that may be important for protein complex formation on nuclear membrane. Furthermore, we demonstrated for the first time that LUMA can interact with another nuclear membrane protein, SUN2, in addition to emerin. Cells expressing mutant LUMA revealed reduced nuclear staining with or without aggregates of emerin and SUN2 together with a higher proportion of abnormally shaped nuclei. In vivo expression of mutant LUMA by electroporation in mouse tibialis anterior muscles likewise demonstrated the decreased staining of emerin and SUN2 on myonuclei. Interpretation: Our results suggest that mutant LUMAs may be associated with EDMD‐related myopathy. ANN NEUROL 2011

The complex of Vacuolar Protein Sorting 34 and 15 (Vps34 and Vps15) has Class III phosphatidylinositol 3-kinase activity and putative roles in nutrient sensing, mammalian Target Of Rapamycin (mTOR) activation by amino acids, cell growth, vesicular trafficking and autophagy. Contrary to expectations, here we show that Vps15-deficient mouse tissues are competent for LC3-positive autophagosome formation and maintain mTOR activation. However, an impaired lysosomal function in mutant cells is traced by accumulation of adaptor protein p62, LC3 and Lamp2 positive vesicles, which can be reverted to normal levels after ectopic overexpression of Vps15. Mice lacking Vps15 in skeletal muscles, develop a severe myopathy. Distinct from the autophagy deficient Atg7(-/-) mutants, pathognomonic morphological hallmarks of autophagic vacuolar myopathy (AVM) are observed in Vps15(-/-) mutants, including elevated creatine kinase plasma levels, accumulation of autophagosomes, glycogen and sarcolemmal features within the fibres. Importantly, Vps34/Vps15 overexpression in myoblasts of Danon AVM disease patients alleviates the glycogen accumulation. Thus, the activity of the Vps34/Vps15 complex is critical in disease conditions such as AVMs, and possibly a variety of other lysosomal storage diseases.

Skeletal muscle contains two distinct stem/progenitor populations. One is the satellite cell, which acts as a muscle stem cell, and the other is the mesenchymal progenitor, which contributes to muscle pathogeneses such as fat infiltration and fibrosis. Detailed and accurate characterization of these progenitors in humans remains elusive. Here, we performed comprehensive cell-surface protein profiling of the two progenitor populations residing in human skeletal muscle and identified three previously unrecognized markers: CD82 and CD318 for satellite cells and CD201 for mesenchymal progenitors. These markers distinguish myogenic and mesenchymal progenitors, and enable efficient isolation of the two types of progenitors. Functional study revealed that CD82 ensures expansion and preservation of myogenic progenitors by suppressing excessive differentiation, and CD201 signaling favors adipogenesis of mesenchymal progenitors. Thus, cell-surface proteins identified here are not only useful markers but also functionally important molecules, and provide valuable insight into human muscle biology and diseases.

Abstract Mutations in amphiphysin‐2/ BIN 1, dynamin 2, and myotubularin are associated with centronuclear myopathy ( CNM ), a muscle disorder characterized by myofibers with atypical central nuclear positioning and abnormal triads. Mis‐splicing of amphiphysin‐2/ BIN 1 is also associated with myotonic dystrophy that shares histopathological hallmarks with CNM . How amphiphysin‐2 orchestrates nuclear positioning and triad organization and how CNM ‐associated mutations lead to muscle dysfunction remains elusive. We find that N‐ WASP interacts with amphiphysin‐2 in myofibers and that this interaction and N‐ WASP distribution are disrupted by amphiphysin‐2 CNM mutations. We establish that N‐ WASP functions downstream of amphiphysin‐2 to drive peripheral nuclear positioning and triad organization during myofiber formation. Peripheral nuclear positioning requires microtubule/Map7/Kif5b‐dependent distribution of nuclei along the myofiber and is driven by actin and nesprins. In adult myofibers, N‐ WASP and amphiphysin‐2 are only involved in the maintenance of triad organization but not in the maintenance of peripheral nuclear positioning. Importantly, we confirmed that N‐ WASP distribution is disrupted in CNM and myotonic dystrophy patients. Our results support a role for N‐ WASP in amphiphysin‐2‐dependent nuclear positioning and triad organization and in CNM and myotonic dystrophy pathophysiology.

Duchenne and Becker muscular dystrophies (DMD/BMD) are the most common inherited neuromuscular disease. The genetic diagnosis is not easily made because of the large size of the dystrophin gene, complex mutational spectrum and high number of tests patients undergo for diagnosis. Multiplex ligation-dependent probe amplification (MLPA) has been used as the initial diagnostic test of choice. Although MLPA can diagnose 70% of DMD/BMD patients having deletions/duplications, the remaining 30% of patients with small mutations require further analysis, such as Sanger sequencing. We applied a high-throughput method using Ion Torrent next-generation sequencing technology and diagnosed 92% of patients with DMD/BMD in a single analysis. We designed a multiplex primer pool for DMD and sequenced 67 cases having different mutations: 37 with deletions/duplications and 30 with small mutations or short insertions/deletions in DMD, using an Ion PGM sequencer. The results were compared with those from MLPA or Sanger sequencing. All deletions were detected. In contrast, 50% of duplications were correctly identified compared with the MLPA method. Small insertions in consecutive bases could not be detected. We estimated that Ion Torrent sequencing could diagnose ~92% of DMD/BMD patients according to the mutational spectrum of our cohort. Our results clearly indicate that this method is suitable for routine clinical practice providing novel insights into comprehensive genetic information for future molecular therapy.

Identification of antisynthetase syndrome (ASS) could be challenging due to inaccessibility and technical difficulty of the serology test for the less common non-Jo-1 antibodies. This study aimed to describe ASS antibody-specific myopathology and evaluate the diagnostic utility of myofiber HLA-DR expression. We reviewed 212 ASS muscle biopsies and compared myopathologic features among subtypes. Additionally, we compared their HLA-DR staining pattern with 602 non-ASS myositis and 140 genetically confirmed myopathies known to have an inflammatory component. We used t-test and Fisher's exact for comparisons and used sensitivity, specificity, positive and negative predictive values to assess the utility of HLA-DR expression for ASS diagnosis. RNAseq performed from a subset of myositis cases and histologically normal muscle biopsies was used to evaluate interferon (IFN)-signaling pathway-related genes. Anti-OJ ASS showed prominent myopathology with higher scores in muscle fiber (4.6 ± 2.0 vs. 2.8 ± 1.8, p = 0.001) and inflammatory domains (6.8 ± 3.2 vs. 4.5 ± 2.9, p = 0.006) than non-OJ ASS. HLA-DR expression and IFN-γ-related genes upregulation were prominent in ASS and inclusion body myositis (IBM). When dermatomyositis and IBM were excluded, HLA-DR expression was 95.4% specific and 61.2% sensitive for ASS with a positive predictive value of 85.9% and a negative predictive value of 84.2%; perifascicular HLA-DR pattern is common in anti-Jo-1 ASS than non-Jo-1 ASS (63.1% vs. 5.1%, p < 0.0001). In the appropriate clinicopathological context, myofiber HLA-DR expression help support ASS diagnosis. The presence of HLA-DR expression suggests involvement of IFN-γ in the pathogenesis of ASS, though the detailed mechanisms have yet to be elucidated.

<b><i>Article abstract</i></b> Thirty-six of 43 maternally related members of a large African American family experienced hearing loss. A muscle biopsy specimen from the proband showed cytochrome <i>c</i> oxidase (COX)-deficient fibers but no ragged-red fibers; biochemical analysis showed marked reduction of COX activity. A novel T7511C point mutation in the tRNA^Ser(UCN) gene was present in almost homoplasmic levels (&gt;95%) in the blood of 18 of 20 family members, and was also found in lower abundance in the other two. Single-fiber PCR showed that the mutational load was greater in COX-deficient muscle fibers. The tRNA^Ser(UCN) gene may be a “hot spot” for mutations associated with maternally transmitted hearing loss.

We report three heterozygous missense mutations of the skeletal muscle alpha actin gene (ACTA1) in three unrelated cases of congenital fiber type disproportion (CFTD) from Japan and Australia. This represents the first genetic cause of CFTD to be identified and confirms that CFTD is genetically heterogeneous. The three mutations we have identified Leucine221Proline, Aspartate292Valine, and Proline332Serine are novel. They have not been found previously in any cases of nemaline, actin, intranuclear rod, or rod-core myopathy caused by mutations in ACTA1. It remains unclear why these mutations cause type 1 fiber hypotrophy but no nemaline bodies. The three mutations all lie on one face of the actin monomer on the surface swept by tropomyosin during muscle activity, which may suggest a common pathological mechanism. All three CFTD cases with ACTA1 mutations had severe congenital weakness and respiratory failure without ophthalmoplegia. There were no clinical features specific to CFTD cases with ACTA1 mutations, but the presence of normal eye movements in a severe CFTD patient may be an important clue for the presence of a mutation in ACTA1.

Distal myopathy with rimmed vacuoles is an autosomal recessive muscle disease with preferential involvement of the tibialis anterior that spares the quadriceps muscles in young adulthood. In a Japanese patient with distal myopathy with rimmed vacuoles, we identified pathogenic mutations in the gene encoding the bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase, which catalyzes the initial two steps in the biosynthesis of sialic acid. In this study, we demonstrated the relationship between the genetic mutations and enzymatic activities using an <i>in vitro</i> expression assay system. Furthermore, we also showed that the levels of sialic acid in muscle and primary cultured cells from DMRV patients were reduced to 60–75% of control. The reactivities to lectins were also variable in some myofibers, suggesting that hyposialylation and abnormal glycosylation in muscles may contribute to the focal accumulations of autophagic vacuoles, amyloid deposits, or both in patient muscle tissue. The addition of ManNAc and NeuAc to primary cultured cells normalized sialylation levels, thus demonstrating the therapeutic potential of these compounds for this disease.

Muscle–eye–brain disease (MEB), an autosomal recessive disorder prevalent in Finland, is characterized by congenital muscular dystrophy, brain malformation and ocular abnormalities. Since the MEB phenotype overlaps substantially with those of Fukuyama-type congenital muscular dystrophy (FCMD) and Walker–Warburg syndrome (WWS), these three diseases are thought to result from a similar pathomechanism. Recently, we showed that MEB is caused by mutations in the protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) gene. We describe here the identification of seven novel disease-causing mutations in six of not only non-Finnish Caucasian but also Japanese and Korean patients with suspected MEB, severe FCMD or WWS. Including six previously reported mutations, the 13 disease-causing mutations we have found thus far are dispersed throughout the entire POMGnT1 gene. We also observed a slight correlation between the location of the mutation and clinical severity in the brain: patients with mutations near the 5′ terminus of the POMGnT1 coding region show relatively severe brain symptoms such as hydrocephalus, while patients with mutations near the 3′ terminus have milder phenotypes. Our results indicate that MEB may exist in population groups outside of Finland, with a worldwide distribution beyond our expectations, and that the clinical spectrum of MEB is broader than recognized previously. These findings emphasize the importance of considering MEB and searching for POMGnT1 mutations in WWS or other congenital muscular dystrophy patients worldwide.

Background Malignant hyperthermia (MH) is a disorder of calcium homeostasis in skeletal muscle triggered by volatile anesthetics or succinylcholine in susceptible persons. More than 100 mutations in the ryanodine receptor type 1 gene (RYR1) have been associated with MH susceptibility, central core disease, or both. RYR1 mutations may account for up to 70% of MH-susceptible cases. The authors aimed to determine the frequency and distribution of RYR1 mutations in the Japanese MH-susceptible population. Methods The authors selected 58 unrelated Japanese diagnosed as MH-susceptible for having an enhanced Ca-induced Ca release rate from the sarcoplasmic reticulum on chemically skinned muscle fibers. They sequenced the entire RYR1 coding region from genomic DNA. Muscle pathology was also characterized. Results Seven previously reported and 26 unknown RYR1 potentially pathogenic sequence variations were identified in 33 patients (56.9%). Of these patients, 48% had cores on muscle biopsy. The mutation detection rate was higher in patients with clear enhancement of Ca-induced Ca release rate (72.4%), whereas all patients with central core disease had RYR1 mutations. Six patients harbored potentially causative compound heterozygous sequence variations. Conclusions Distribution and frequency of RYR1 mutations differed markedly from those of the North American and European MH-susceptible population. Comprehensive screening of the RYR1 gene is recommended for molecular investigations in MH-susceptible individuals, because many mutations are located outside the "hot spots." Based on the observed occurrence of compound heterozygous state, the prevalence of a possibly predisposing phenotype in the Japanese population might be as high as 1 in 2,000 people.

Autophagic vacuoles are a frequent feature in numerous neuromuscular disorders. However, they are also pathognomonic morphologic hallmarks in a slowly emerging new group of conditions called autophagic vacuolar myopathies (AVMs), of which Danon disease, originally called “lysosomal glycogen storage disease with normal acid maltase,” is the best known entity. Other such conditions, often although not always described from Japan, are X-linked myopathy with excessive authophagy, infantile autophagic vacuolar myopathy, adult-onset autophagic vacuolar myopathy with multiorgan involvement, and X-linked congenital autophagic vacuolar myopathy. Although only 1 protein, the transmembranous lysosomal protein LAMP-2, has been found mutated in Danon disease, the remaining AVMs are genetically still incompletely identified. Several of these conditions not only share autophagic vacuoles, but such autophagic vacuoles also have morphologic properties of the sarcolemma, thus rendering them autophagic vacuoles with sarcolemmal features, an almost pathognomonic phenomenon of this group of disorders.

The present nomenclature of the splice variants of the lysosome-associated membrane protein type 2 (LAMP-2) is confusing. The LAMP-2a isoform is uniformly named in human, chicken, and mouse, but the LAMP-2b and LAMP-2c isoforms are switched in human as compared with mouse and chicken. We propose to change the nomenclature of the chicken and mouse b and c isoforms to agree with that currently used for the human isoforms. To avoid confusion in the literature, we further propose to adopt the use of capital letters for the updated nomenclature of all the isoforms in all three species: LAMP-2A, LAMP-2B, and LAMP-2C.

To determine the frequency of primary collagen VI deficiency in congenital muscular dystrophy (CMD) in Japan and to establish the genotype-phenotype correlation.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.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.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.

Peroxisome proliferator-activated receptor-gamma co-activator-1alpha (PGC-1alpha) is a key nuclear receptor co-activator for mitochondrial biogenesis. Here we report that overexpression of PGC-1alpha in skeletal muscles increased mitochondrial number and caused atrophy of skeletal muscle, especially type 2B fiber-rich muscles (gastrocnemius, quadriceps, and plantaris). Muscle atrophy became evident at 25 weeks of age, and a portion of the muscle was replaced by adipocytes. Mice showed increased energy expenditure and reduced body weight; thyroid hormone levels were normal. Mitochondria exhibited normal respiratory chain activity per mitochondrion; however, mitochondrial respiration was not inhibited by an ATP synthase inhibitor, oligomycin, clearly indicating that oxidative phosphorylation was uncoupled. Accordingly, ATP content in gastrocnemius was markedly reduced. A similar phenotype is observed in Luft's disease, a mitochondrial disorder that involves increased uncoupling of respiration and muscle atrophy. Our results indicate that overexpression of PGC-1alpha in skeletal muscle increases not only mitochondrial biogenesis but also uncoupling of respiration, resulting in muscle atrophy.

Filamin C is an actin-crosslinking protein that is specifically expressed in cardiac and skeletal muscles. Although mutations in the filamin C gene cause human myopathy with cardiac involvement, the function of filamin C in vivo is not yet fully understood. Here we report a medaka mutant, zacro (zac), that displayed an enlarged heart, caused by rupture of the myocardiac wall, and progressive skeletal muscle degeneration in late embryonic stages. We identified zac to be a homozygous nonsense mutation in the filamin C (flnc) gene. The medaka filamin C protein was found to be localized at myotendinous junctions, sarcolemma, and Z-disks in skeletal muscle, and at intercalated disks in the heart. zac embryos showed prominent myofibrillar degeneration at myotendinous junctions, detachment of myofibrils from sarcolemma and intercalated disks, and focal Z-disk destruction. Importantly, the expression of γ-actin, which we observed to have a strong subcellular localization at myotendinous junctions, was specifically reduced in zac mutant myotomes. Inhibition of muscle contraction by anesthesia alleviated muscle degeneration in the zac mutant. These results suggest that filamin C plays an indispensable role in the maintenance of the structural integrity of cardiac and skeletal muscles for support against mechanical stress.

Dynamin 2 (DNM2) is a large GTPase implicated in many cellular functions, including cytoskeleton regulation and endocytosis. Although ubiquitously expressed, DNM2 was found mutated in two genetic disorders affecting different tissues: autosomal dominant centronuclear myopathy (ADCNM; skeletal muscle) and peripheral Charcot-Marie-Tooth neuropathy (peripheral nerve). To gain insight into the function of DNM2 in skeletal muscle and the pathological mechanisms leading to ADCNM, we introduced wild-type DNM2 (WT-DNM2) or R465W DNM2 (RW-DNM2), the most common ADCNM mutation, into adult wild-type mouse skeletal muscle by intramuscular adeno-associated virus injections. We detected altered localization of RW-DNM2 in mouse muscle. Several ADCNM features were present in RW-DNM2 mice: fiber atrophy, nuclear mislocalization, and altered mitochondrial staining, with a corresponding reduction in specific maximal muscle force. The sarcomere and triad structures were also altered. We report similar findings in muscle biopsy specimens from an ADCNM patient with the R465W mutation. In addition, expression of wild-type DNM2 induced some muscle defects, albeit to a lesser extent than RW-DNM2, suggesting that the R465W mutation has enhanced activity in vivo. In conclusion, we show the RW-DNM2 mutation acts in a dominant manner to cause ADCNM in adult muscle, and the disease arises from a primary defect in skeletal muscle rather than secondary to peripheral nerve involvement. Therefore, DNM2 plays important roles in the maintenance of adult muscle fibers. Dynamin 2 (DNM2) is a large GTPase implicated in many cellular functions, including cytoskeleton regulation and endocytosis. Although ubiquitously expressed, DNM2 was found mutated in two genetic disorders affecting different tissues: autosomal dominant centronuclear myopathy (ADCNM; skeletal muscle) and peripheral Charcot-Marie-Tooth neuropathy (peripheral nerve). To gain insight into the function of DNM2 in skeletal muscle and the pathological mechanisms leading to ADCNM, we introduced wild-type DNM2 (WT-DNM2) or R465W DNM2 (RW-DNM2), the most common ADCNM mutation, into adult wild-type mouse skeletal muscle by intramuscular adeno-associated virus injections. We detected altered localization of RW-DNM2 in mouse muscle. Several ADCNM features were present in RW-DNM2 mice: fiber atrophy, nuclear mislocalization, and altered mitochondrial staining, with a corresponding reduction in specific maximal muscle force. The sarcomere and triad structures were also altered. We report similar findings in muscle biopsy specimens from an ADCNM patient with the R465W mutation. In addition, expression of wild-type DNM2 induced some muscle defects, albeit to a lesser extent than RW-DNM2, suggesting that the R465W mutation has enhanced activity in vivo. In conclusion, we show the RW-DNM2 mutation acts in a dominant manner to cause ADCNM in adult muscle, and the disease arises from a primary defect in skeletal muscle rather than secondary to peripheral nerve involvement. Therefore, DNM2 plays important roles in the maintenance of adult muscle fibers. Dynamins are large, multifunctional GTPase proteins that were initially identified as microtubule-binding proteins1Shpetner H.S. Vallee R.B. Identification of dynamin, a novel mechanochemical enzyme that mediates interactions between microtubules.Cell. 1989; 59: 421-432Abstract Full Text PDF PubMed Scopus (341) Google Scholar and more recently were recognized to play an important role in actin cytoskeleton assembly2Unsworth K.E. Mazurkiewicz P. Senf F. Zettl M. McNiven M. Way M. Holden D.W. Dynamin is required for F-actin assembly and pedestal formation by enteropathogenic Escherichia coli (EPEC).Cell Microbiol. 2007; 9: 438-449Crossref PubMed Scopus (38) Google Scholar and membrane trafficking and endocytosis.3Jones S.M. Howell K.E. Henley J.R. Cao H. McNiven M.A. Role of dynamin in the formation of transport vesicles from the trans-Golgi network.Science. 1998; 279: 573-577Crossref PubMed Scopus (274) Google Scholar, 4van der Bliek A.M. Meyerowitz E.M. Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic.Nature. 1991; 351: 411-414Crossref PubMed Scopus (592) Google Scholar Dynamins can form polymerized rings around membrane tubules,5Takei K. McPherson P.S. Schmid S.L. De Camilli P. Tubular membrane invaginations coated by dynamin rings are induced by GTP-gamma S in nerve terminals.Nature. 1995; 374: 186-190Crossref PubMed Scopus (654) Google Scholar and the mechanism by which dynamins regulate membrane fission has been intensively studied.6Roux A. Uyhazi K. Frost A. De Camilli P. GTP-dependent twisting of dynamin implicates constriction and tension in membrane fission.Nature. 2006; 441: 528-531Crossref PubMed Scopus (389) Google Scholar, 7Pucadyil T.J. Schmid S.L. Real-time visualization of dynamin-catalyzed membrane fission and vesicle release.Cell. 2008; 135: 1263-1275Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar Dynamins contain an N-terminal GTPase domain, middle domain, Pleckstrin homology (PH) domain (phosphoinositide binding), GTPase effector domain (GED), and a Proline-rich domain (PRD) for protein-protein interactions. Dynamin 1 is expressed specifically in neurons, dynamin 3 is expressed mainly in the brain and testis, and dynamin 2 (DNM2) is ubiquitously expressed. Despite its ubiquitous expression, mutations in DNM2 induce tissue-specific diseases; missense mutations in the middle and C-terminal part of the PH domain lead to autosomal dominant centronuclear myopathy (ADCNM; OMIM 160150), which affects skeletal muscle,8Bitoun M. Maugenre S. Jeannet P.Y. Lacene E. Ferrer X. Laforet P. Martin J.J. Laporte J. Lochmuller H. Beggs A.H. Fardeau M. Eymard B. Romero N.B. Guicheney P. Mutations in dynamin 2 cause dominant centronuclear myopathy.Nat Genet. 2005; 37: 1207-1209Crossref PubMed Scopus (333) Google Scholar whereas mutations in the N-terminal part of the PH domain are linked to dominant Charcot-Marie-Tooth (OMIM 606482) peripheral neuropathy.9Zuchner S. Noureddine M. Kennerson M. Verhoeven K. Claeys K. De Jonghe P. Merory J. Oliveira S.A. Speer M.C. Stenger J.E. Walizada G. Zhu D. Pericak-Vance M.A. Nicholson G. Timmerman V. Vance J.M. Mutations in the pleckstrin homology domain of dynamin 2 cause dominant intermediate Charcot-Marie-Tooth disease.Nat Genet. 2005; 37: 289-294Crossref PubMed Scopus (287) Google Scholar Although studies on DNM2 have been performed mainly in cultured cells, the function of DNM2 in skeletal muscle has barely been studied. Moreover, mechanisms of the tissue-specific impact of DNM2 mutations leading to myopathy are not characterized, and it is not clear whether DNM2 mutations have an impact on muscle development or adult muscle maintenance. Centronuclear myopathies (CNMs) are congenital myopathies characterized by muscle weakness associated with fiber atrophy, predominance of type I fibers, and increased centralization of nuclei not secondary to muscle regeneration.10Jungbluth H. Wallgren-Pettersson C. Laporte J. Centronuclear (myotubular) myopathy.Orphanet J Rare Dis. 2008; 3: 26Crossref PubMed Scopus (217) Google Scholar DNM2-related CNM commonly presents as an adult-onset, mild form of CNM8Bitoun M. Maugenre S. Jeannet P.Y. Lacene E. Ferrer X. Laforet P. Martin J.J. Laporte J. Lochmuller H. Beggs A.H. Fardeau M. Eymard B. Romero N.B. Guicheney P. Mutations in dynamin 2 cause dominant centronuclear myopathy.Nat Genet. 2005; 37: 1207-1209Crossref PubMed Scopus (333) Google Scholar; however, cases of severe neonatal onset have been identified.11Bitoun M. Bevilacqua J.A. Prudhon B. Maugenre S. Taratuto A.L. Monges S. Lubieniecki F. Cances C. Uro-Coste E. Mayer M. Fardeau M. Romero N.B. Guicheney P. Dynamin 2 mutations cause sporadic centronuclear myopathy with neonatal onset.Ann Neurol. 2007; 62: 666-670Crossref PubMed Scopus (115) Google Scholar In addition to the autosomal dominant form, the X-linked form or myotubular myopathy (OMIM 310400) is due to mutations in the phosphoinositides phosphatase myotubularin,12Laporte J. Hu L.J. Kretz C. Mandel J.L. Kioschis P. Coy J.F. Klauck S.M. Poustka A. Dahl N. A gene mutated in X-linked myotubular myopathy defines a new putative tyrosine phosphatase family conserved in yeast.Nat Genet. 1996; 13: 175-182Crossref PubMed Scopus (513) Google Scholar and several autosomal recessive CNM patients (OMIM 160150) are linked to mutations in the membrane remodeling protein amphiphysin 2 (BIN1).13Nicot A.S. Toussaint A. Tosch V. Kretz C. Wallgren-Pettersson C. Iwarsson E. Kingston H. Garnier J.M. Biancalana V. Oldfors A. Mandel J.L. Laporte J. Mutations in amphiphysin 2 (BIN1) disrupt interaction with dynamin 2 and cause autosomal recessive centronuclear myopathy.Nat Genet. 2007; 39: 1134-1139Crossref PubMed Scopus (280) Google Scholar Rare sporadic cases of CNM have also been identified with variants in the ryanodine receptor (RyR1)14Jungbluth H. Zhou H. Sewry C.A. Robb S. Treves S. Bitoun M. Guicheney P. Buj-Bello A. Bonnemann C. Muntoni F. Centronuclear myopathy due to a de novo dominant mutation in the skeletal muscle ryanodine receptor (RYR1) gene.Neuromuscul Disord. 2007; 17: 338-345Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 15Wilmshurst J.M. Lillis S. Zhou H. Pillay K. Henderson H. Kress W. Muller C.R. Ndondo A. Cloke V. Cullup T. Bertini E. Boennemann C. Straub V. Quinlivan R. Dowling J.J. Al-Sarraj S. Treves S. Abbs S. Manzur A.Y. Sewry C.A. Muntoni F. Jungbluth H. RYR1 mutations are a common cause of congenital myopathies with central nuclei.Ann Neurol. 2010; 68: 717-726Crossref PubMed Scopus (198) Google Scholar or in the myotubularin-related protein hJUMPY/MTMR14.16Tosch V. Rohde H.M. Tronchere H. Zanoteli E. Monroy N. Kretz C. Dondaine N. Payrastre B. Mandel J.L. Laporte J. A novel PtdIns3P and PtdIns(3,5)P2 phosphatase with an inactivating variant in centronuclear myopathy.Hum Mol Genet. 2006; 15: 3098-3106Crossref PubMed Scopus (108) Google Scholar In this study, we expressed DNM2 containing the most common ADCNM mutation, R465W, in adult wild-type skeletal muscle by intramuscular adeno-associated virus (AAV) injections. We induced several features of CNM, and we have compared these findings with muscle biopsy specimens from a patient with the same mutation. We show that DNM2 is important in maintenance of adult muscle fibers and that this DNM2 mutation acts in a dominant manner to perturb DNM2 function. Full-length human isoform DNM2 cDNA was purchased from Geneservice (DNAFORM, Kanagawa, Japan) (IMAGE clone 5722134, GenBank accession number NM BC039596) and was cloned into pENTR1A (Invitrogen, Carlsbad, CA) and then recombined into a pAAV-MCS vector using the Gateway system, with or without a C-terminal green fluorescent protein (GFP) fusion tag. The R465W mutation was introduced by primer-directed PCR mutagenesis. All constructs were verified by sequencing. pXR1 (AAV1) plasmid was a gift from Jude Samulski at the Gene Therapy Center, the University of North Carolina at Chapel Hill. Informed consent was obtained from human subjects. The patient with a DNM2 Arg465Trp mutation will be reported in more detail elsewhere. Primary antibodies used were mouse anti–α-actinin (EA-53; Sigma-Aldrich, St. Louis, MO), DHPRα1 (Cav1.1) subunit (MA3-920; Affinity Bioreagents, Golden, CO), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; MAB374; Chemicon, Temecula, CA), and desmin (Y-20; Santa Cruz Biotechnology, Santa Cruz, CA). Rabbit anti-DNM2 antibodies (2680 and 2865) were made onsite at the polyclonal antibody facility of the Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC) (described below). Rabbit anti-dystrophin antibody was a gift from Karim Hnia and Dominique Mornet, ERI25, Montpellier, France. Alexa-conjugated secondary antibodies were from Invitrogen. Secondary antibodies against mouse and rabbit IgG, conjugated with horseradish peroxidase (HRP), were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). An ECL chemiluminescent reaction kit was purchased from Pierce (Rockford, IL). Hoechst was purchased from Sigma-Aldrich (B2883). The AAV Helper-Free system was purchased from Stratagene (La Jolla, CA) (catalog number 240071). Two DNM2 rabbit antibodies were generated: R2680, raised against the linker between the PH and GED domains of DNM2 (peptide EKDQAENEDGAQENTF), and R2865, raised against the PRD of DNM2 (peptide HSPTPQRRPVSSVHPPGRPPAVR). Serum samples were purified on peptide-coupled SulfoLink columns (Pierce) (R2680) or with the Imject Maleimide Activated Mariculture KLH kit (INTERCHIM SA, Montlucon, France) (R2865). DNM2 antibodies were validated for immunoblotting in COS-1 cells transfected with human DNM2 using peptide competition and validated for immunofluorescence in mouse muscle sections using peptide competition and showed similar results. AAV2/1 vectors were generated by a triple transfection of AAV-293 cell line with pAAV2 insert containing the insert under the control of the CMV promoter and flanked by serotype 2 inverted terminal repeats, pXR1 containing rep and cap genes of AAV serotype 1, and pHelper encoding the adenovirus helper functions. Cell lysates were subjected to 3 freeze/thaw cycles, then treated with 50 U/mL of Benzonase (Sigma) for 30 minutes at 37°C, and clarified by centrifugation. Viral vectors were purified by Iodixanol gradient ultracentrifugation followed by dialysis and concentration against Dulbecco's Phosphate Buffered Saline using centrifugal filters (Amicon Ultra-15 Centrifugal Filter Devices 30K, Millipore, Bedford). Physical particles were quantified by real-time PCR using a plasmid standard pAAV-eGFP, and titers are expressed as viral genomes per milliliter (vg/mL). rAAV titers used in these experiments were 5 to 7 × 1011 vg/mL. Five- to 6-week-old, male, wild-type, 129PAS mice were anesthetized by i.p. injection of 5 μL/g of ketamine (20 mg/mL; Virbac, Carros, France) and xylazine (0.4%, Rompun; Bayer, Wuppertal, Germany). Tibialis anterior (TA) muscles were injected with 25 μL of AAV2/1 preparations or sterile PBS solution. Animals were housed in a temperature-controlled room (19°C to 22°C) with a 12:12-hour light/dark cycle. Mice were sacrificed by CO2 inhalation followed by cervical dislocation, according to national and European legislations on animal experimentation. The TA muscles were dissected [with the mice under anesthesia when required for transmission electron microscopy (TEM)] 2 to 4 weeks post injection (PI) and frozen in nitrogen-cooled isopentane and liquid nitrogen for histologic and immunoblot assays, respectively. In mice injected i.m. with GFP-AAV2/1, we observed a transduction efficiency of 75% to 100%, which is comparable to previously published results.17Louboutin J.P. Wang L. Wilson J.M. Gene transfer into skeletal muscle using novel AAV serotypes.J Gene Med. 2005; 7: 442-451Crossref PubMed Scopus (96) Google Scholar Longitudinal and transverse cryosections (8 μm) or semithin (500 nm) sections of mouse TA skeletal muscles and human muscle were prepared, fixed, and stained with antibodies to DHPRα1 (1:500), desmin (1:500), α-actinin (1:1000), R2680-DNM2 (1:500), R2865-DNM2 (1:500), and dystrophin (1:400). Nuclei were detected by costaining with Hoechst (Sigma-Aldrich) for 10 minutes. Sections were viewed using a laser scanning confocal microscope (TCS SP2; Leica Microsystems, Sunnyvale, CA). Intensity of staining (Figure 1B) was measured using the plot profile function in ImageJ analysis software (W.S. Rasband, ImageJ, National Institutes of Health, Bethesda, MD, 1998–2009; http://rsb.info.nih.gov/ij). Alternatively, air-dried transverse sections were fixed and stained with hematoxylin-eosin (HE), succinate dehydrogenase (SDH) or NADH-tetrazolium reductase (TR) activity and viewed with a fluorescence microscope (DM4000; Leica Microsystems). The cross-sectional area was analyzed in HE sections from TA mouse skeletal muscle, using the software MetaMorph (Molecular Devices). The cross-sectional area was calculated (>500 fibers per mouse) from three to five mice per group. The percentage of TA muscle fibers with centralized or internalized nuclei was counted in more than 400 fibers in at least three mice using the cell counter plugin in ImageJ image analysis software. The TA muscle was dissected from the hind limb and fixed in 4% paraformaldehyde in PBS for 30 minutes. Muscles were then incubated with 0.1 mol/L glycine in PBS for 30 minutes, followed by 30% sucrose/PBS solution overnight at 4°C. Muscle were washed with PBS, and fibers were then isolated under a binocular microscope. Fibers were then permeabilized and stained with DNM2 and α-actinin antibodies and viewed by confocal microscopy. Mice were anesthetized by intraperitoneal injection of 10 μL/g of ketamine (20 mg/mL; Virbac) and xylazine (0.4%, Rompun; Bayer). Muscle biopsy specimens from hind limbs were fixed with 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer (pH 7.2) and processed as described.18Buj-Bello A. Laugel V. Messaddeq N. Zahreddine H. Laporte J. Pellissier J.F. Mandel J.L. The lipid phosphatase myotubularin is essential for skeletal muscle maintenance but not for myogenesis in mice.Proc Natl Acad Sci U S A. 2002; 99: 15060-15065Crossref PubMed Scopus (172) Google Scholar For T-tubule analysis, the T-tubule shape factor was measured by manually outlining the shape of the T-tubule and using the circularity measurement in ImageJ image analysis software. On average, 50 T-tubules per mouse for two mice per group were measured. Potassium ferrocyanide staining was performed as described previously.19Al-Qusairi L. Weiss N. Toussaint A. Berbey C. Messaddeq N. Kretz C. Sanoudou D. Beggs A.H. Allard B. Mandel J.L. Laporte J. Jacquemond V. Buj-Bello A. T-tubule disorganization and defective excitation-contraction coupling in muscle fibers lacking myotubularin lipid phosphatase.Proc Natl Acad Sci U S A. 2009; 106: 18763-18768Crossref PubMed Scopus (142) Google Scholar Muscle force measurements were evaluated by measuring in situ muscle contraction in response to nerve and muscle stimulation, as described previously.20Vignaud A. Cebrian J. Martelly I. Caruelle J.P. Ferry A. Effect of anti-inflammatory and antioxidant drugs on the long-term repair of severely injured mouse skeletal muscle.Exp Physiol. 2005; 90: 487-495Crossref PubMed Scopus (32) Google Scholar, 21Vignaud A. Hourde C. Medja F. Agbulut O. Butler-Browne G. Ferry A. Impaired skeletal muscle repair after ischemia-reperfusion injury in mice.J Biomed Biotechnol. 2010; 2010: 724914Crossref PubMed Scopus (50) Google Scholar Briefly, animals were anesthetized (i.p., pentobarbital sodium, 50 mg × kg−1). The distal tendon of the TA was detached and tied with a silk ligature to an isometric transducer (Harvard Bioscience, Holliston, MA). The sciatic nerve was distally stimulated, response to tetanic stimulation (pulse frequency of 50 to 143 Hz) was recorded, and absolute maximal force was determined. After contractile measurements, the animals were sacrificed by cervical dislocation. To determine specific maximal force, TA muscles were dissected and weighted. Muscles were then stored as described for further analysis. Total, soluble, and insoluble proteins were extracted from the skeletal muscle of mice. Mouse TA muscle (stored at −80°C before use) was minced and homogenized on ice for 3 × 30 seconds (Ultra Thurax homogenizer) in 10 times the w/v of 1% NP-40 Tris-Cl buffer (pH 8) then extracted for 30 minutes at 4°C, and used for Western blotting. For preparation of soluble and insoluble fractions, lysates were then centrifuged at 8000 × g for 5 minutes, and the soluble fraction (supernatant) and insoluble fraction (pellet solubilized in 8 mol/L urea) were collected. Protein concentration was determined using a DC protein assay kit (Bio-Rad Laboratories, Hercules, CA) and lysates analyzed by SDS–polyacrylamide gel electrophoresis and Western blotting (nitrocellulose membrane). Primary antibodies used were R2865-DNM2 (1:1000) and GAPDH (1:10,000); secondary antibodies were anti-rabbit HRP or anti-mouse HRP. Western blot films were scanned and band signal intensities were determined using ImageJ software. Densitometry values were expressed as a fold difference relative to the control, standardized to corresponding total GAPDH values. All microscopy was performed at the Imaging Centre of the IGBMC. All samples for microscopy were mounted in Fluorsave reagent (Merck, Summit, NJ) and viewed at room temperature. Confocal microscopy was performed using a confocal laser scanning microscope (TCS SP2; Leica Microsystems) on a DMRXA2 upright microscope. Fluorescence and light microscopy was performed using a fluorescence microscope (DM4000; Leica Microsystems) fitted with a color CCD camera (Coolsnap cf color; Photometrics, Tucson, AZ) camera. Metamorph software (Molecular Devices) and ImageJ analysis software were used for image analysis. Statistical analysis was performed using the unpaired Student's t-test unless stated otherwise. P < 0.05 was considered significant. DNM2 is ubiquitously expressed in human tissues22Cook T.A. Urrutia R. McNiven M.A. Identification of dynamin 2, an isoform ubiquitously expressed in rat tissues.Proc Natl Acad Sci U S A. 1994; 91: 644-648Crossref PubMed Scopus (163) Google Scholar, 23Diatloff-Zito C. Gordon A.J. Duchaud E. Merlin G. Isolation of an ubiquitously expressed cDNA encoding human dynamin II, a member of the large GTP-binding protein family.Gene. 1995; 163: 301-306Crossref PubMed Scopus (28) Google Scholar; however, the localization of DNM2 in skeletal muscle has not been well characterized. In mouse isolated muscle fibers, DNM2 colocalized with the Z-line marker α-actinin, as observed by immunofluorescence (Figure 1A) and confirmed by analyzing the intensity of staining of DNM2 and α-actinin, which displayed a consistent overlapping profile at the Z-line (Figure 1B). In mouse skeletal muscle sections, desmin, a different Z-line marker, colocalized with DNM2, although this was slightly more discontinuous compared with the colocalization observed with α-actinin. In contrast, DHPRα, which labels T-tubules, formed a doublet band that did not colocalize but aligned in close proximity with DNM2 staining (Figure 1C). Therefore, in mouse striated muscle DNM2 appears to localize to the Z-line, in close proximity to the T-tubules. DNM2 localization in human skeletal muscle was consistent with that observed in mouse muscle. In neonatal human muscle (1.5 months old), DNM2 formed a transverse banding pattern consistent with Z-line staining, whereas DHPRα was located in a longitudinal arrangement (Figure 2A), indicating that DNM2 localizes to the sarcomeric unit before the final maturation of T-tubules to a transverse position. In adult human muscle the DNM2 localization was consistent with Z-line striations on longitudinal sections (Figure 2C, top left), whereas on transverse sections an intricate network of DNM2 staining was observed within the fiber, with a more dense localization at the subsarcolemmal region (Figure 2C, bottom left). Therefore, DNM2 is recruited to the sarcomeric unit before the maturation of T-tubules is complete and localizes to the Z-line in mature muscle. The most common mutation found in patients with ADCNM is an arginine to tryptophan missense at position 465 (R465W), located within the middle domain of DNM2 (RW-DNM2) (Figure 2B).8Bitoun M. Maugenre S. Jeannet P.Y. Lacene E. Ferrer X. Laforet P. Martin J.J. Laporte J. Lochmuller H. Beggs A.H. Fardeau M. Eymard B. Romero N.B. Guicheney P. Mutations in dynamin 2 cause dominant centronuclear myopathy.Nat Genet. 2005; 37: 1207-1209Crossref PubMed Scopus (333) Google Scholar, 24Susman R.D. Quijano-Roy S. Yang N. Webster R. Clarke N.F. Dowling J. Kennerson M. Nicholson G. Biancalana V. Ilkovski B. Flanigan K.M. Arbuckle S. Malladi C. Robinson P. Vucic S. Mayer M. Romero N.B. Urtizberea J.A. Garcia-Bragado F. Guicheney P. Bitoun M. Carlier R.Y. North K.N. Expanding the clinical, pathological and MRI phenotype of DNM2-related centronuclear myopathy.Neuromuscul Disord. 2010; 20: 229-237Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar Patients with this mutation exhibit the three typical CNM features: atrophic fibers, centralized and internalized nuclei, and abnormal oxidative staining with the appearance of radial sarcoplasmic strands (RSSs) on NADH-TR staining (Figure 2D).25Romero N.B. Centronuclear myopathies: a widening concept.Neuromuscul Disord. 2010; 20: 223-228Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar In muscle sections from an RW-DNM2 patient, DNM2 localized as a striated pattern on longitudinal sections, although this pattern appeared disturbed and more irregular (Figure 2C, top right). On transversal sections, DNM2 was present but the intricate network of staining appeared perturbed (Figure 2C, bottom right). Altogether, this suggests that the RW-DNM2 mutation is linked to abnormal protein localization and muscle intracellular organization. Complete deletion of DNM2 in mouse resulted in embryonic lethality,26Ferguson S.M. Raimondi A. Paradise S. Shen H. Mesaki K. Ferguson A. Destaing O. Ko G. Takasaki J. Cremona O. O'Toole E. De Camilli P. Coordinated actions of actin and BAR proteins upstream of dynamin at endocytic clathrin-coated pits.Dev Cell. 2009; 17: 811-822Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar and RW-DNM2 heterozygous knock-in mice exhibit a mild impairment in skeletal muscle structure, with skeletal muscle atrophy and a corresponding reduction in force detected.27Durieux A.C. Vignaud A. Prudhon B. Viou M.T. Beuvin M. Vassilopoulos S. Fraysse B. Ferry A. Laine J. Romero N.B. Guicheney P. Bitoun M. A centronuclear myopathy-dynamin 2 mutation impairs skeletal muscle structure and function in mice.Hum Mol Genet. 2010; 19: 4820-4836Crossref PubMed Scopus (92) Google Scholar However, a strong CNM phenotype was not observed in these mice, and no increase in central or internalized nuclei was detected. It is currently unknown whether the CNM phenotype is due to a haploinsufficiency of DNM2 expression or function or whether the mutant protein acts in a dominant manner. In addition, whether the disease results from the altered function of DNM2 in muscle development or in adult muscle maintenance is not clear. Moreover, it was not clear whether the muscle phenotype is due to a primary impairment of peripheral nerves because some DNM2 mutations cause a peripheral neuropathy. To decipher these three points, we used an original in vivo approach to attempt to recreate ADCNM in wild-type adult mouse skeletal muscle and to investigate the normal function of DNM2 in wild-type muscle. Expression of DNM2 containing the R465W mutation was achieved by intramuscular injections of an AAV cognate vector into the TA muscle of wild-type 5- to 6-week-old mice, an age where the muscle mass is nearly fully developed. This allowed the role of DNM2 to be studied specifically in adult skeletal muscle. Mice were injected with WT-DNM2 or RW-DNM2 with a C-terminal GFP tag or GFP alone as a control. The injection of WT-DNM2, while acting as a control for RW-DNM2–injected muscle, also allowed us to investigate the function of WT-DNM2 in muscle. The addition of a GFP tag allowed the differentiation between AAV-transduced and endogenous DNM2 expression. Untagged DNM2 constructs were also used and gave comparable results as described below. The level of DNM2 protein expression was determined using Western blot analysis and densitometry. In both WT-DNM2– and RW-DNM2–transduced muscles a fivefold increase in total DNM2 expression was observed compared with GFP-transduced and noninjected muscles (see Supplemental Figure S1, A and B, at http://ajp.amjpathol.org). No significant difference in protein solubility was observed between exogenous WT-DNM2 or RW-DNM2 and endogenous DNM2 (see Supplemental Figure S1, C and D, at http://ajp.amjpathol.org), indicating AAV-DNM2 expression did not alter DNM2 association to membranes. Therefore, full-length AAV-DNM2 is expressed in mouse skeletal muscle. Because full-length RW-DNM2 was efficiently expressed, we next determined whether any features of ADCNM were exhibited in this model. At 2 weeks PI, mice transduced with RW-DNM2 exhibited a slight but significant reduction in TA muscle mass, and by 4 weeks PI the muscles were reduced to almost 50% the weight of GFP control and PBS-injected muscles (Figure 3A). To confirm that the decrease in muscle mass was due to muscle fiber atrophy, the cross-sectional area of muscle fibers was determined from transverse sections stained with HE (Figure 3, D and F). A decrease in fiber area was observed in RW-DNM2 transduced TA muscle both 2 weeks (Figure 3, B and D) and 4 weeks PI (Figure 3, B and F), corresponding with the decrease in muscle mass. The decrease in average muscle fiber area was due to an increase in atrophic fibers compared with WT-DNM2 and GFP-transduced muscle (Figure 3C), confirming RW-DNM2 transduction into wild-type muscle induced muscle fiber atrophy. WT-DNM2 also exhibited a slight but significant decrease in muscle weight and fiber size, however to a significantly lesser extent than RW-DNM2 muscle. It is possible that the level of endogenous DNM2 expression needs to be tightly regulated and that overexpression of WT-DNM2 causes some perturbations in muscle size. Healthy muscle fibers contain nuclei beneath the sarcolemma, and a hallmark of DNM2-related CNM is the large increase in fibers containing internal or central nuclei (Figure 2D).10Jungbluth H. Wallgren-Pettersson C. Laporte J. Centronuclear (myotubular) myopathy.Orphanet J Rare Dis. 2008; 3: 26Crossref PubMed Scopus (217) Google Scholar, 25Romero N.B. Centronuclear myopathies: a

Common fragile sites (CFSs) are specific chromosome regions that exhibit an increased frequency of breaks when cells are exposed to a DNA-replication inhibitor such as aphidicolin. PARK2 and DMD, the causative genes for autosomal-recessive juvenile Parkinsonism and Duchenne and Becker muscular dystrophy, respectively, are two very large genes that are located within aphidicolin-induced CFSs. Gross rearrangements within these two genes are frequently observed as the causative mutations for these diseases, and similar alterations within the large fragile sites that surround these genes are frequently observed in cancer cells. To elucidate the molecular mechanisms underlying this fragility, we performed a custom-designed high-density comparative genomic hybridization analysis to determine the junction sequences of approximately 500 breakpoints in germ cell lines and cancer cell lines involving PARK2 or DMD. The sequence signatures where these breakpoints occur share some similar features both in germ cell lines and in cancer cell lines. Detailed analyses of these structures revealed that microhomologies are predominantly involved in rearrangement processes. Furthermore, breakpoint-clustering regions coincide with the latest-replicating region and with large nuclear-lamina-associated domains and are flanked by the highest-flexibility peaks and R/G band boundaries, suggesting that factors affecting replication timing collectively contribute to the vulnerability for rearrangement in both germ cell and somatic cell lines. Common fragile sites (CFSs) are specific chromosome regions that exhibit an increased frequency of breaks when cells are exposed to a DNA-replication inhibitor such as aphidicolin. PARK2 and DMD, the causative genes for autosomal-recessive juvenile Parkinsonism and Duchenne and Becker muscular dystrophy, respectively, are two very large genes that are located within aphidicolin-induced CFSs. Gross rearrangements within these two genes are frequently observed as the causative mutations for these diseases, and similar alterations within the large fragile sites that surround these genes are frequently observed in cancer cells. To elucidate the molecular mechanisms underlying this fragility, we performed a custom-designed high-density comparative genomic hybridization analysis to determine the junction sequences of approximately 500 breakpoints in germ cell lines and cancer cell lines involving PARK2 or DMD. The sequence signatures where these breakpoints occur share some similar features both in germ cell lines and in cancer cell lines. Detailed analyses of these structures revealed that microhomologies are predominantly involved in rearrangement processes. Furthermore, breakpoint-clustering regions coincide with the latest-replicating region and with large nuclear-lamina-associated domains and are flanked by the highest-flexibility peaks and R/G band boundaries, suggesting that factors affecting replication timing collectively contribute to the vulnerability for rearrangement in both germ cell and somatic cell lines.

Choline kinase is the first step enzyme for phosphatidylcholine (PC) de novo biosynthesis. Loss of choline kinase activity in muscle causes rostrocaudal muscular dystrophy (rmd) in mouse and congenital muscular dystrophy in human, characterized by distinct mitochondrial morphological abnormalities. We performed biochemical and pathological analyses on skeletal muscle mitochondria from rmd mice. No mitochondria were found in the center of muscle fibers, while those located at the periphery of the fibers were significantly enlarged. Muscle mitochondria in rmd mice exhibited significantly decreased PC levels, impaired respiratory chain enzyme activities, decreased mitochondrial ATP synthesis, decreased coenzyme Q and increased superoxide production. Electron microscopy showed the selective autophagic elimination of mitochondria in rmd muscle. Molecular markers of mitophagy, including Parkin, PINK1, LC3, polyubiquitin and p62, were localized to mitochondria of rmd muscle. Quantitative analysis shows that the number of mitochondria in muscle fibers and mitochondrial DNA copy number were decreased. We demonstrated that the genetic defect in choline kinase in muscle results in mitochondrial dysfunction and subsequent mitochondrial loss through enhanced activation of mitophagy. These findings provide a first evidence for a pathomechanistic link between de novo PC biosynthesis and mitochondrial abnormality.

Mutations in LMNA cause wide variety of disorders including Emery-Dreifuss muscular dystrophy, limb girdle muscular dystrophy, and congenital muscular dystrophy. We recently found a LMNA mutation in a patient who was previously diagnosed as infantile onset inflammatory myopathy. In this study, we screened for LMNA mutations in 20 patients suspected to have inflammatory myopathy with onset at 2years or younger. The diagnosis of inflammatory myopathy was based on muscle pathology with presence of perivascular cuffing and/or endomysial/perimysial lymphocyte infiltration. We identified heterozygous LMNA mutations in 11 patients (55%), who eventually developed joint contractures and/or cardiac involvement after the infantile period. Our findings suggest that LMNA mutation should be considered in myopathy patients with inflammatory changes during infancy, and that this may help avoid life-threatening events associated with laminopathy.

We ascertained a nuclear family in which three of four siblings were affected with an unclassified autosomal recessive myopathy characterized by severe weakness, respiratory impairment, scoliosis, joint contractures, and an unusual combination of dystrophic and myopathic features on muscle biopsy. Whole genome sequence from one affected subject was filtered using linkage data and variant databases. A single gene, MEGF10, contained nonsynonymous mutations that co-segregated with the phenotype. Affected subjects were compound heterozygous for missense mutations c.976T > C (p.C326R) and c.2320T > C (p.C774R). Screening the MEGF10 open reading frame in 190 patients with genetically unexplained myopathies revealed a heterozygous mutation, c.211C > T (p.R71W), in one additional subject with a similar clinical and histological presentation as the discovery family. All three mutations were absent from at least 645 genotyped unaffected control subjects. MEGF10 contains 17 atypical epidermal growth factor-like domains, each of which contains eight cysteine residues that likely form disulfide bonds. Both the p.C326R and p.C774R mutations alter one of these residues, which are completely conserved in vertebrates. Previous work showed that murine Megf10 is required for preserving the undifferentiated, proliferative potential of satellite cells, myogenic precursors that regenerate skeletal muscle in response to injury or disease. Here, knockdown of megf10 in zebrafish by four different morpholinos resulted in abnormal phenotypes including unhatched eggs, curved tails, impaired motility, and disorganized muscle tissue, corroborating the pathogenicity of the human mutations. Our data establish the importance of MEGF10 in human skeletal muscle and suggest satellite cell dysfunction as a novel myopathic mechanism.

The recessively inherited, adult onset, quadriceps sparing myopathy with a predilection for distal muscles has received multiple historic names. The disorder was described in 1981 in Japanese patients and termed Nonaka Distal Myopathy [ [1] Nonaka I. Sunohara N. Ishiura S. Satoyoshi E. Familial distal myopathy with rimmed vacuole and lamellar (myeloid) body formation. J Neurol Sci. 1981; 51: 141-155 Abstract Full Text PDF PubMed Scopus (250) Google Scholar ], later commonly referred to as Distal Myopathy with Rimmed Vacuoles (DMRV) (OMIM#605820). In 1984, the disorder was described as vacuolar myopathy sparing the quadriceps in Iranian-Jewish patients [ [2] Argov Z. Yarom R. “Rimmed vacuole myopathy” sparing the quadriceps. A unique disorder in Iranian Jews. J Neurol Sci. 1984; 64: 33-43 Abstract Full Text PDF PubMed Scopus (228) Google Scholar ], later commonly referred to as Inclusion Body Myopathy 2 (IBM2) or Hereditary Inclusion Body Myopathy (HIBM) (OMIM#600737). Mapping of the causative gene to the same locus on chromosome 9 in different cohorts of patient [ 3 Mitrani-Rosenbaum S. Argov Z. Blumenfeld A. Seidman C.E. Seidman J.G. Hereditary inclusion body myopathy maps to chromosome 9p1-q1. Hum Mol Genet. 1996; 5: 159-163 Crossref PubMed Scopus (89) Google Scholar , 4 Ikeuchi T. Asaka T. Saito M. et al. Gene locus for autosomal recessive distal myopathy with rimmed vacuoles maps to chromosome 9. Ann Neurol. 1997; 41: 432-437 Crossref PubMed Scopus (84) Google Scholar ], and ultimately identification of mutations in the causative gene GNE in all cohorts [ 5 Eisenberg I. Avidan N. Potikha T. et al. The UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene is mutated in recessive hereditary inclusion body myopathy. Nat Genet. 2001; 29: 83-87 Crossref PubMed Scopus (433) Google Scholar , 6 Nishino I. Noguchi S. Murayama K. et al. Distal myopathy with rimmed vacuoles is allelic to hereditary inclusion body myopathy. Neurology. 2002; 59: 1689-1693 Crossref PubMed Scopus (192) Google Scholar ], confirmed that these myopathies are in fact the same condition.

<h3>Objective:</h3> To clarify whether there is any association between inclusion body myositis (IBM) and hepatitis C virus (HCV) infection. <h3>Methods:</h3> We assessed the prevalence of HCV infection in 114 patients with IBM whose muscle biopsies were analyzed pathologically for diagnostic purpose from 2002 to 2012 and in 44 age-matched patients with polymyositis diagnosed in the same period as a control by administering a questionnaire survey to the physicians in charge. We also compared clinicopathologic features including the duration from onset to development of representative symptoms of IBM and the extent of representative pathologic changes between patients with IBM with and without HCV infection. <h3>Results:</h3> A significantly higher number of patients with IBM (28%) had anti-HCV antibodies as compared with patients with polymyositis (4.5%; odds ratio 8.2, 95% confidence interval 1.9–36) and the general Japanese population in their 60s (3.4%). Furthermore, between patients with IBM with and without HCV infection, we did not find any significant difference in the clinicopathologic features, indicating that the 2 groups have essentially the same disease regardless of HCV infection. <h3>Conclusion:</h3> Our results provide the statistical evidence for an association between IBM and HCV infection, suggesting a possible pathomechanistic link between the 2 conditions.

Sir, The papers by Mescam-Mancini et al. (2015) and Stenzel et al. (2015) are of particular interest, at least partially addressing the important question whether anti-synthetase syndrome is pathologically distinct from other idiopathic inflammatory myopathies. Mescam-Mancini et al. (2015) beautifully demonstrated that patients with anti-Jo-1 antibodies, one of the anti-aminoacyl-tRNA synthetase (ARS) antibodies, characteristically show perifascicular necrosis on muscle pathology. However, they did not deal with anti-synthetase syndrome patients with anti-ARS antibodies other than anti-Jo-1, raising a question whether perifascicular necrosis is characteristic only of myopathies associated with anti-Jo-1 antibodies or is observed also in those associated with other anti-ARS antibodies. We have analysed muscle samples from patients with anti-Jo-1 antibodies ( n …

Of 207 adult patients with idiopathic inflammatory myopathies, detection of autoantibodies by RNA immunoprecipitation showed that 99 patients (48%) were antibody-positive. We divided these 99 into five subgroups: anti-signal recognition particle (SRP), anti-aminoacyl transfer RNA synthetase, anti-Ku, anti-U1RNP, and anti-SSA/B. Younger age at onset, severe weakness, muscle atrophy, elevated creatine kinase, and necrosis in muscle fibers without inflammatory cell infiltration were found significantly more frequently among the patients with anti-SRP antibodies (n=41) compared to the antibody-negative patients (n=108). Autoantibody detection by RNA immunoprecipitation can provide useful information associated with clinical and histological findings.

We describe four families with affected siblings showing unique clinical features: early-onset (before 1 year of age) progressive diffuse brain atrophy with regression, postnatal microcephaly, postnatal growth retardation, muscle weakness/atrophy, and respiratory failure. By whole-exome sequencing, we identified biallelic TBCD mutations in eight affected individuals from the four families. TBCD encodes TBCD (tubulin folding co-factor D), which is one of five tubulin-specific chaperones playing a pivotal role in microtubule assembly in all cells. A total of seven mutations were found: five missense mutations, one nonsense, and one splice site mutation resulting in a frameshift. In vitro cell experiments revealed the impaired binding between most mutant TBCD proteins and ARL2, TBCE, and β-tubulin. The in vivo experiments using olfactory projection neurons in Drosophila melanogaster indicated that the TBCD mutations caused loss of function. The wide range of clinical severity seen in this neurodegenerative encephalopathy may result from the residual function of mutant TBCD proteins. Furthermore, the autopsied brain from one deceased individual showed characteristic neurodegenerative findings: cactus and somatic sprout formations in the residual Purkinje cells in the cerebellum, which are also seen in some diseases associated with mitochondrial impairment. Defects of microtubule formation caused by TBCD mutations may underlie the pathomechanism of this neurodegenerative encephalopathy.

Missense variants in RNA-binding proteins (RBPs) underlie a spectrum of disease phenotypes, including amyotrophic lateral sclerosis, frontotemporal dementia, and inclusion body myopathy. Here, we present ten independent families with a severe, progressive muscular dystrophy, reminiscent of oculopharyngeal muscular dystrophy (OPMD) but of much earlier onset, caused by heterozygous frameshift variants in the RBP hnRNPA2/B1. All disease-causing frameshift mutations abolish the native stop codon and extend the reading frame, creating novel transcripts that escape nonsense-mediated decay and are translated to produce hnRNPA2/B1 protein with the same neomorphic C-terminal sequence. In contrast to previously reported disease-causing missense variants in HNRNPA2B1, these frameshift variants do not increase the propensity of hnRNPA2 protein to fibrillize. Rather, the frameshift variants have reduced affinity for the nuclear import receptor karyopherin β2, resulting in cytoplasmic accumulation of hnRNPA2 protein in cells and in animal models that recapitulate the human pathology. Thus, we expand the phenotypes associated with HNRNPA2B1 to include an early-onset form of OPMD caused by frameshift variants that alter its nucleocytoplasmic transport dynamics.

Valosin-containing protein (VCP) disease, caused by mutations in the VCP gene, results in myopathy, Paget's disease of bone (PBD) and frontotemporal dementia (FTD). Natural history and genotype-phenotype correlation data are limited. This study characterises patients with mutations in VCP gene and investigates genotype-phenotype correlations.Descriptive retrospective international study collecting clinical and genetic data of patients with mutations in the VCP gene.Two hundred and fifty-five patients (70.0% males) were included in the study. Mean age was 56.8±9.6 years and mean age of onset 45.6±9.3 years. Mean diagnostic delay was 7.7±6 years. Symmetric lower limb weakness was reported in 50% at onset progressing to generalised muscle weakness. Other common symptoms were ventilatory insufficiency 40.3%, PDB 28.2%, dysautonomia 21.4% and FTD 14.3%. Fifty-seven genetic variants were identified, 18 of these no previously reported. c.464G>A (p.Arg155His) was the most frequent variant, identified in the 28%. Full time wheelchair users accounted for 19.1% with a median time from disease onset to been wheelchair user of 8.5 years. Variant c.463C>T (p.Arg155Cys) showed an earlier onset (37.8±7.6 year) and a higher frequency of axial and upper limb weakness, scapular winging and cognitive impairment. Forced vital capacity (FVC) below 50% was as risk factor for being full-time wheelchair user, while FVC <70% and being a full-time wheelchair user were associated with death.This study expands the knowledge on the phenotypic presentation, natural history, genotype-phenotype correlations and risk factors for disease progression of VCP disease and is useful to improve the care provided to patient with this complex disease.

Adult-onset idiopathic inflammatory myopathy (IIM) is associated with an increased cancer risk within the 3 years preceding and following IIM onset. Evidence- and consensus-based recommendations for IIM-associated cancer screening can potentially improve outcomes. This International Guideline for IIM-Associated Cancer Screening provides recommendations addressing IIM-associated cancer risk stratification, cancer screening modalities and screening frequency. The international Expert Group formed a total of 18 recommendations via a modified Delphi approach using a series of online surveys. First, the recommendations enable an individual patient's IIM-associated cancer risk to be stratified into standard, moderate or high risk according to the IIM subtype, autoantibody status and clinical features. Second, the recommendations outline a 'basic' screening panel (including chest radiography and preliminary laboratory tests) and an 'enhanced' screening panel (including CT and tumour markers). Third, the recommendations advise on the timing and frequency of screening via basic and enhanced panels, according to risk status. The recommendations also advise consideration of upper or lower gastrointestinal endoscopy, nasoendoscopy and 18F-FDG PET–CT scanning in specific patient populations. These recommendations are aimed at facilitating earlier IIM-associated cancer detection, especially in those who are at a high risk, thus potentially improving outcomes, including survival. In this Evidence-Based Guideline article, an international, multidisciplinary group of experts presents evidence-based consensus recommendations on screening for cancer in patients with adult-onset idiopathic inflammatory myopathy, addressing cancer risk stratification, screening modalities and screening frequency.

α-Dystroglycan is a component of the dystrophin-glycoprotein-complex, which is the major mechanism of attachment between the cytoskeleton and the extracellular matrix. Muscle–eye–brain disease (MEB) is an autosomal recessive disorder characterized by congenital muscular dystrophy, ocular abnormalities and lissencephaly. We recently found that MEB is caused by mutations in the protein O-linked mannose β1,2-N-acetylglucosaminyltransferase (POMGnT1) gene. POMGnT1 is a glycosylation enzyme that participates in the synthesis of O-mannosyl glycan, a modification that is rare in mammals but is known to be a laminin-binding ligand of α-dystroglycan. Here we report a selective deficiency of α-dystroglycan in MEB patients. This finding suggests that α-dystroglycan is a potential target of POMGnT1 and that altered glycosylation of α-dystroglycan may play a critical role in the pathomechanism of MEB and some forms of muscular dystrophy.

Depletion and multiple deletions of mitochondrial DNA (mtDNA) have been associated with a growing number of autosomal diseases that have been classified as defects of intergenomic communication. MNGIE, an autosomal recessive disorder associated with mtDNA alterations is due to mutations in thymidine phosphorylase that may cause imbalance of the mitochondrial nucleotide pool. Subsequently, mutations in the mitochondrial proteins adenine nucleotide translocator 1, Twinkle, and polymerase gamma have been found to cause autosomal dominant progressive external ophthalmoplegia with multiple deletions of mtDNA. Uncovering the molecular bases of intergenomic communication defects will enhance our understanding of the mechanisms responsible for maintaining mtDNA integrity.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is caused by mutations in the gene encoding thymidine phosphorylase (TP). The clinical manifestations of MNGIE are recognizable and homogeneous, but in the early stages, the disease is often misdiagnosed. This study assesses the reliability of biochemical assays to diagnose MNGIE.We studied 180 patients with clinical features suggestive of MNGIE, 14 asymptomatic TP mutation carriers, and 20 controls. TP enzyme activity in the buffy coat was determined by a fixed-time method, and the plasma nucleosides thymidine (dThd) and deoxyuridine (dUrd) were assessed by a gradient-elution reversed phase HPLC method. TP was sequenced through standard procedures in patients who met the clinical criteria for MNGIE.Twenty-five of the 180 patients fulfilled the clinical criteria for MNGIE and had homozygous or compound heterozygous TP mutations. All had drastically decreased TP activity [mean (SD), 10 (15) nmol thymine formed. h(-1). (mg protein)(-1) vs 634 (217) nmol thymine formed. h(-1). (mg protein)(-1) for the controls]. Relative to the control mean, TP activities were reduced to 35% in mutation carriers and 65% in MNGIE-like patients. All 25 MNGIE patients had detectable plasma dThd [8.6 (3.4) micromol/L] and dUrd [14.2 (4.4) micromol/L]. Controls, carriers, and MNGIE-like patients showed no detectable plasma dThd and dUrd.We propose a diagnostic algorithm based on the determination of plasma dThd and dUrd, TP activity in buffy coat, or both to make a definitive diagnosis of MNGIE. Increased concentrations of dThd (>3 micromol/L) and dUrd (>5 micromol/L) in plasma or a decrease in buffy coat TP activity to </=8% relative to controls is sufficient to diagnose MNGIE.

Myocyte enhancer factor 2 (MEF2) plays essential roles in transcriptional control of muscle development. However, signaling pathways acting downstream of MEF2 are largely unknown. Here, we performed a microarray analysis using Mef2c-null mouse embryos and identified a novel MEF2-regulated gene encoding a muscle-specific protein kinase, Srpk3, belonging to the serine arginine protein kinase (SRPK) family, which phosphorylates serine/arginine repeat-containing proteins. The Srpk3 gene is specifically expressed in the heart and skeletal muscle from embryogenesis to adulthood and is controlled by a muscle-specific enhancer directly regulated by MEF2. Srpk3-null mice display a new entity of type 2 fiber-specific myopathy with a marked increase in centrally placed nuclei; while transgenic mice overexpressing Srpk3 in skeletal muscle show severe myofiber degeneration and early lethality. We conclude that normal muscle growth and homeostasis require MEF2-dependent signaling by Srpk3.

Emery-Dreifuss muscular dystrophy is an inherited muscular disorder clinically characterized by slowly progressive weakness affecting humero-peroneal muscles, early joint contractures, and cardiomyopathy with conduction block. The X-linked recessive form is caused by mutation in the EMD gene encoding an integral protein of the inner nuclear membrane, emerin. In this study, mutant mice lacking emerin were produced by insertion of a neomycin resistance gene into exon 6 of the coding gene. Tissues taken from mutant mice lacked emerin. The mutant mice displayed a normal growth rate indistinguishable from their littermates and were fertile. No marked muscle weakness or joint abnormalities were observed; however, rotarod test revealed altered motor coordination. Electrocardiography showed mild prolongation of atrioventricular conduction time in emerin-lacking male mice older than 40 weeks of age. Electron microscopic analysis of skeletal and cardiac muscles from emerin-lacking mice revealed small vacuoles, which mostly bordered the myonuclei. Our results suggest that emerin deficiency causes minimal motor and cardiac dysfunctions in mice with a structural fragility of myonuclei.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) syndrome is a rare, multisystem disorder characterized clinically by ptosis, progressive external ophthalmoplegia, gastrointestinal dysmotility, leukoencephalopathy, thin body habitus, and myopathy. Laboratory studies reveal defects of oxidative-phosphorylation and multiple mtDNA deletions frequently in skeletal muscle. We studied four ethnically distinct families affected with this apparently autosomal recessive disorder. Probands from each family were shown, by Southern blot, to have multiple mtDNA deletions in skeletal muscle. We mapped the MNGIE locus to 22q13.32-qter, distal to D22S1161, with a maximum two-point LOD score of 6.80 at locus D22S526. Cosegregation of MNGIE with a single chromosomal region in families with diverse ethnic backgrounds suggests that we have mapped an important locus for this disorder. We found no evidence to implicate three candidate genes in this region, by using direct sequence analysis for DNA helicase II and by assaying enzyme activities for arylsulfatase A and carnitine palmitoyltransferase.

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.

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

We examined 136 patients with mitochondrial DNA (mtDNA) deletion. Clinical diagnoses included chronic progressive external ophthalmoplegia (94 patients); Kearns-Sayre syndrome (KSS; 33 patients); Pearson's marrow-pancreas syndrome (six patients); and Leigh syndrome, Reye-like syndrome, and mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (one patient). The length and location of deletion were highly variable. Only one patient had deletion within the so-called shorter arc between the two origins of mtDNA replication. The length of deletion and the number of deleted transfer ribonucleic acid (tRNAs) showed a significant relationship with age at onset. Furthermore, KSS patients had longer and larger numbers of deleted tRNAs, which could be risk factors for the systemic involvement of single mtDNA deletion diseases. We found 81 patterns of deletion. Direct repeats of 4 bp or longer flanking the breakpoints were found in 96 patients (70.5%) and those of 10 bp or longer in 49 patients (36.0%). We found two other common deletions besides the most common deletion (34 patients: 25.0%): the 2,310-bp deletion from nt 12113 to nt 14421 (11 patients: 8.0%) and the 7,664-bp deletion from nt 6330 to nt 13993 (ten patients: 7.3%). These deletions had incomplete direct repeats longer than 13 bp with one base mismatch.

Causative genes have been identified only in four types of lipid storage myopathies (LSMs): SLC22A5 for primary carnitine deficiency (PCD); ETFA, ETFB, and ETFDH for multiple acyl-coenzyme A dehydrogenation deficiency (MADD); PNPLA2 for neutral lipid storage disease with myopathy (NLSDM); and ABHD5 for neutral lipid storage disease with ichthyosis. However, the frequency of these LSMs has not been determined. We found mutations in only 9 of 37 LSM patients (24%): 3 in SLC22A5; 4 in MADD-associated genes; and 2 in PNPLA2. This low frequency suggests the existence of other causative genes. Muscle coenzyme Q(10) levels were normal or only mildly reduced in two MADD patients, indicating that ETFDH mutations may not always be associated with CoQ(10) deficiency. The 2 patients with PNPLA2 mutations had progressive, non-episodic muscle disease with rimmed vacuoles. This suggests there is a different pathomechanism from other LSMs.

Mutations in SCO2, a protein required for the proper assembly and functioning of cytochrome c oxidase (COX; complex IV of the mitochondrial respiratory chain), cause a fatal infantile cardioencephalomyopathy with COX deficiency. We have generated mice harboring a Sco2 knock-out (KO) allele and a Sco2 knock-in (KI) allele expressing an E-->K mutation at position 129 (E129K), corresponding to the E140K mutation found in almost all human SCO2-mutated patients. Whereas homozygous KO mice were embryonic lethals, homozygous KI and compound heterozygous KI/KO mice were viable, but had muscle weakness; biochemically, they had respiratory chain deficiencies as well as complex IV assembly defects in multiple tissues. There was a concomitant reduction in mitochondrial copper content, but the total amount of copper in examined tissues was not reduced. These mouse models should be of use in further studies of Sco2 function, as well as in testing therapeutic approaches to treat the human disorder.

Lysosomal myopathies are hereditary myopathies characterized morphologically by the presence of autophagic vacuoles. In mammals, autophagy plays an important role for the turnover of cellular components, particularly in response to starvation or glucagons. In normal muscle, autolysosomes or autophagosomes are typically inconspicuous. In distinct neuromuscular disorders, however, lysosomes become structurally abnormal and functionally impaired, leading to the accumulation of autophagic vacuoles in myofibers. In some instances, the accumulation of autophagic vacuoles can be a prominent feature, implicating autophagy as a contributor to disease pathomechanism and/or progression. At present, there are two disorders in the muscle that are associated with a primary defect in lysosomal proteins, namely Pompe disease and Danon disease. This review will give a brief discussion on these disorders, highlighting the role of autophagy in disease progression.

<b>Background: </b> 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 (<i>RYR1</i>) mutations in the C-terminal domain had type 1 fibers, nearly exclusively, in addition to typical central cores. <b>Objective: </b> To investigate whether CNMDU1 is associated with <i>RYR1</i> mutation. <b>Methods: </b> 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 <i>RYR1</i>. <b>Results: </b> Four of 10 patients had a heterozygous mutation, three missense and one deletion, all in the C-terminal domain of <i>RYR1</i>. Two missense mutations were previously reported in CCD patients. Clinically, patients with mutations in <i>RYR1</i> showed milder phenotype compared with those without mutations. <b>Conclusion: </b> Congenital neuromuscular disease with uniform type 1 fiber (CNMDU1) in 40% of patients is associated with mutations in the C-terminal domain of <i>RYR1</i>, suggesting that CNMDU1 is allelic to central core disease at least in some patients.

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.

Collagen VI is widely distributed throughout extracellular matrices (ECMs) in various tissues. In skeletal muscle, collagen VI is particularly concentrated in and adjacent to basement membranes of myofibers. Ullrich congenital muscular dystrophy (UCMD) is caused by mutations in either <i>COL6A1</i>, <i>COL6A2</i> or <i>COL6A3</i> gene, thereby leading to collagen VI deficiency in the ECM. It is known to occur through either recessive or dominant genetic mechanism, the latter most typically by <i>de novo</i> mutations. UCMD is well defined by the clinicopathological hallmarks including distal hyperlaxity, proximal joint contractures, protruding calcanei, scoliosis and respiratory insufficiency. Recent reports have depicted the robust natural history of UCMD; that is, loss of ambulation by early teenage years, rapid decline in respiratory function by 10 years of age and early-onset, rapidly progressive scoliosis. Muscle pathology is characterised by prominent interstitial fibrosis disproportionate to the relative paucity of necrotic and regenerating fibres. To date, treatment for patients is supportive for symptoms such as joint contractures, respiratory failure and scoliosis. There have been clinical trials based on the theory of mitochondrion-mediated myofiber apoptosis or impaired autophagy. Furthermore, the fact that collagen VI producing cells in skeletal muscle are interstitial mesenchymal cells can support proof of concept for stem cell-based therapy.

Currently, clinical trials for new therapeutic strategies are being planned for Duchenne and Becker muscular dystrophies (DMD/BMD). However, it is difficult to obtain adequate numbers of patients in clinical trials. As solutions to these problems, patient registries are an important resource worldwide, especially in rare diseases such as DMD/BMD.We developed a national registry of Japanese DMD/BMD patients in collaboration with TREAT-NMD. The registry includes male Japanese DMD/BMD patients whose genetic status has been confirmed by genetic analysis. The registry includes patients throughout Japan.As of February 2012, 583 DMD and 105 BMD patients were registered. Most individuals aged less than 20 years. In terms of genetic mutations of registrants of DMD and BMD, deletion of exons was the most frequent (61.4% and 79.0%) followed by point mutations (24.5% and 14.3%) and duplications (13.6% and 4.8%), respectively. 43.6% of DMD are capable of walking, and 76.2% of BMD registrants are able to walk. 41.1% of DMD registrants in the database were treated using steroids. 29.5% of DMD and 23.8% of BMD registrants were prescribed one cardiac medicine at least. 22% of DMD used ventilator support, and non-invasive support was common. Small numbers of DMD and BMD registrants, only 3.9% and 1.0% of them, have received scoliosis surgery. 57 (9.8%) patients were eligible to clinical trial focused on 'skipping' exon 51.The Remudy has already demonstrated utility in clinical researches and standardization of patients care for DMD/BMD. This new DMD/BMD patient registry facilitates the synchronization of clinical drug development in Japan with that in other countries.

Patients with GNE myopathy, a progressive and debilitating disease caused by a genetic defect in sialic acid biosynthesis, rely on supportive care and eventually become wheelchair-bound. To elucidate whether GNE myopathy is treatable at a progressive stage of the disease, we examined the efficacy of sialic acid supplementation on symptomatic old GNE myopathy mice that have ongoing, active muscle degeneration. We examined the therapeutic effect of a less metabolized sialic acid compound (6'-sialyllactose) or free sialic acid (N-acetylneuraminic acid) by oral, continuous administration to 50-week-old GNE myopathy mice for 30 weeks. To evaluate effects on their motor performance in living mice, spontaneous locomotion activity on a running wheel was measured chronologically at 50, 65, 72 and 80 weeks of age. The size, force production, and pathology of isolated gastrocnemius muscle were analysed at the end point. Sialic acid level in skeletal muscle was also measured. Spontaneous locomotion activity was recovered in 6'-sialyllactose-treated mice, while NeuAc-treated mice slowed the disease progression. Treatment with 6'-sialyllactose led to marked restoration of hyposialylation in muscle and consequently to robust improvement in the muscle size, contractile parameters, and pathology as compared to NeuAc. This is due to the fact that 6'-sialyllactose is longer working as it is further metabolized to free sialic acid after initial absorption. 6'-sialyllactose ameliorated muscle atrophy and degeneration in symptomatic GNE myopathy mice. Our results provide evidence that GNE myopathy can be treated even at a progressive stage and 6'-sialyllactose has more remarkable advantage than free sialic acid, providing a conceptual proof for clinical use in patients.

DNAJB6, which encodes DnaJ homolog, subfamily B, member 6 (DNAJB6) was recently identified as a causative gene for limb-girdle muscular dystrophy type 1D (LGMD1D). DNAJB6 is a member of heat shock protein 40 and contains a J domain, G/F domain and C-terminal domain. Only three different mutations have been identified in 11 families. In this study, we identified seven Japanese individuals from four unrelated families who carried a DNAJB6 mutation. We found a novel p.Phe96Ile substitution and a previously reported p.Phe96Leu change in the G/F domain of DNAJB6. All affected individuals showed slowly progressive muscle weakness, mainly in their legs, and their muscle pathology showed cytoplasmic inclusions and rimmed vacuoles. Our immunohistochemical analysis detected cytoplasmic accumulations associated with chaperone-assisted selective autophagy together with intranuclear accumulations of DNAJB6 and heat shock 22-kD protein 8 (HSPB8). This is the first report of Asian patients with LGMD1D. Our new findings may contribute to understanding the pathological mechanisms of this myopathy.

Transport protein particle (TRAPP) is a multiprotein complex involved in endoplasmic reticulum-to-Golgi trafficking. Zebrafish with a mutation in the TRAPPC11 orthologue showed hepatomegaly with steatosis and defects in visual system development. In humans, TRAPPC11 mutations have been reported in only three families showing limb-girdle muscular dystrophy (LGMD) or myopathy with movement disorders and intellectual disability.We screened muscular dystrophy genes using next-generation sequencing and performed associated molecular and biochemical analyses in a patient with fatty liver and cataract in addition to infantile-onset muscle weakness.We identified the first Asian patient with TRAPPC11 mutations. Muscle pathology demonstrated typical dystrophic changes and liver biopsy revealed steatosis. The patient carried compound heterozygous mutations of a previously reported missense and a novel splice-site mutation. The splice-site change produced two aberrantly-spliced transcripts that were both predicted to result in translational frameshift and truncated proteins. Full-length TRAPPC11 protein was undetectable on immunoblotting.This report widens the phenotype of TRAPPC11-opathy as the patient showed the following: (1) congenital muscular dystrophy phenotype rather than LGMD; (2) steatosis and infantile-onset cataract, both not observed in previously reported patients; but (3) no ataxia or abnormal movement, clearly indicating that TRAPPC11 plays a physiological role in multiple tissues in human.

Inherited skeletal muscle diseases are genetically heterogeneous diseases caused by mutations in more than 150 genes. This has made it challenging to establish a high-throughput screening method for identifying causative gene mutations in clinical practice.In the present study, we developed a useful method for screening gene mutations associated with the pathogenesis of skeletal muscle diseases.We established four target gene panels, each covering all exonic and flanking regions of genes involved in the pathogenesis of the following muscle diseases: (1) muscular dystrophy (MD), (2) congenital myopathy/congenital myasthenic syndrome, (3) metabolic myopathy and (4) myopathy with protein aggregations/rimmed vacuoles. We assigned one panel to each patient based on the results of clinical and histological analyses of biopsied muscle samples and performed high-throughput sequencing by using Ion PGM next-generation sequencer. We also performed protein analysis to confirm defective proteins in patients with major muscular dystrophies. Further, we performed muscle-derived cDNA analysis to identify splice-site mutations.We identified possible causative gene mutations in 33% of patients (62/188) included in this study. Our results showed that the MD panel was the most useful, with a diagnostic rate of 46.2%.Thus, we developed a high-throughput sequencing technique for diagnosing inherited muscle diseases. The use of this technique along with histological and protein analyses may be useful and cost-effective for screening mutations in patients with inherited skeletal muscle diseases.

Immune-mediated necrotizing myopathy (IMNM), also known as necrotizing autoimmune myopathy, is a histologic entity characterized by marked necrosis in the absence of prominent lymphocytes.1 Risk factors or triggers for IMNM include statin treatment, cancer, and connective tissue disease (CTD).1,2 Although autoantibodies against signal recognition particle (SRP) or 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) are regarded as markers for IMNM, they are not always detected in sera of pathologically defined patients with IMNM. The autoimmune mechanisms of IMNM are not elucidated. Acknowledgment: The authors thank all the physicians who provided muscle biopsy and serum samples and detailed clinical information; and Kaoru Tatezawa and Kazu Iwasawa (Department of Neuromuscular Research, National Institute of Neuroscience, and Department of Genome Medicine Development, Medical Genome Center, National Center of Neurology and Psychiatry) for their technical support.

Recently, de novo mutations in TBL1XR1 were found in two patients with autism spectrum disorders. Here, we report on a Japanese girl presenting with West syndrome, Rett syndrome-like and autistic features. Her initial development was normal until she developed a series of spasms at 5 months of age. Electroencephalogram at 7 months showed a pattern of hypsarrhythmia, which led to a diagnosis of West syndrome. Stereotypic hand movements appeared at 8 months of age, and autistic features such as deficits in communication, hyperactivity and excitability were observed later, at 4 years and 9 months. Whole exome sequencing of the patient and her parents revealed a de novo TBL1XR1 mutation [c.209 G>A (p.Gly70Asp)] occurring at an evolutionarily conserved amino acid in an F-box-like domain. Our report expands the clinical spectrum of TBL1XR1 mutations to West syndrome with Rett-like features, together with autistic features.

To assess long-term (2 years) effects of bimagrumab in participants with sporadic inclusion body myositis (sIBM).Participants (aged 36-85 years) who completed the core study (RESILIENT [Efficacy and Safety of Bimagrumab/BYM338 at 52 Weeks on Physical Function, Muscle Strength, Mobility in sIBM Patients]) were invited to join an extension study. Individuals continued on the same treatment as in the core study (10 mg/kg, 3 mg/kg, 1 mg/kg bimagrumab or matching placebo administered as IV infusions every 4 weeks). The co-primary outcome measures were 6-minute walk distance (6MWD) and safety.Between November 2015 and February 2017, 211 participants entered double-blind placebo-controlled period of the extension study. Mean change in 6MWD from baseline was highly variable across treatment groups, but indicated progressive deterioration from weeks 24-104 in all treatment groups. Overall, 91.0% (n = 142) of participants in the pooled bimagrumab group and 89.1% (n = 49) in the placebo group had ≥1 treatment-emergent adverse event (AE). Falls were slightly higher in the bimagrumab 3 mg/kg group vs 10 mg/kg, 1 mg/kg, and placebo groups (69.2% [n = 36 of 52] vs 56.6% [n = 30 of 53], 58.8% [n = 30 of 51], and 61.8% [n = 34 of 55], respectively). The most frequently reported AEs in the pooled bimagrumab group were diarrhea 14.7% (n = 23), involuntary muscle contractions 9.6% (n = 15), and rash 5.1% (n = 8). Incidence of serious AEs was comparable between the pooled bimagrumab and the placebo group (18.6% [n = 29] vs 14.5% [n = 8], respectively).Extended treatment with bimagrumab up to 2 years produced a good safety profile and was well-tolerated, but did not provide clinical benefits in terms of improvement in mobility. The extension study was terminated early due to core study not meeting its primary endpoint.Clinicaltrials.gov identifier NCT02573467.This study provides Class IV evidence that for patients with sIBM, long-term treatment with bimagrumab was safe, well-tolerated, and did not provide meaningful functional benefit. The study is rated Class IV because of the open-label design of extension treatment period 2.

Characteristics of myositis with anti-Ku antibodies are poorly understood. The purpose of this study was to elucidate the pathologic features of myositis associated with anti-Ku antibodies, compared with immune-mediated necrotizing myopathy (IMNM) with anti-signal recognition particle (SRP) and anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) antibodies, in muscle biopsy-oriented registration cohorts in Japan and Germany.We performed a retrospective pathology review of patients with anti-Ku myositis samples diagnosed in the Japanese and German cohorts. We evaluated histologic features and performed HLA phenotyping.Fifty biopsied muscle samples in the Japanese cohort and 10 in the German cohort were obtained. After exclusion of myositis-specific autoantibodies or other autoimmune connective tissue diseases, 26 samples (43%) of anti-Ku antibody-positive myositis were analyzed. All the samples shared some common features with IMNM, whereas they showed expression of MHC class II and clusters of perivascular inflammatory cells more frequently than the anti-SRP/HMGCR IMNM samples (71% vs 7%/16%; p < 0.005/<0.005; 64% vs 0%/0%; p < 0.005/<0.005). Anti-Ku myositis biopsies could be divided into 2 subgroups based on the extent of necrosis and regeneration. The group with more abundant necrosis and regeneration showed a higher frequency of MHC class II expression and perivascular inflammatory cell clusters. HLA phenotyping in the 44 available patients showed possible associations of HLA-DRB1*03:01, HLA-DRB1*11:01, and HLA-DQB1*03:01 (p = 0.0045, 0.019, and 0.027; odds ratio [OR] 50.2, 4.6, and 2.8; 95% CI 2.6-2942.1, 1.1-14.5, and 1.0-7.0) in the group with less conspicuous necrosis and regeneration. On the contrary, in the group of more abundant necrosis and regeneration, the allele frequencies of HLA-A*24:02, HLA-B*52:01, HLA-C*12:02, and HLA-DRB1*15:02 were lower than those of healthy controls (p = 0.0036, 0.027, 0.016, and 0.026; OR = 0.27, 0, 0, and 0; 95% CI 0.1-0.7, 0-0.8, 0-0.8, and 0-0.8). However, these HLA associations did not remain significant after statistical correction for multiple testing.While anti-Ku myositis shows necrotizing myopathy features, they can be distinguished from anti-SRP/HMGCR IMNM by their MHC class II expression and clusters of perivascular inflammatory cells. The HLA analyses suggest that anti-Ku myositis may have different subsets associated with myopathologic subgroups.

Bethlem myopathy (OMIM # 158810) is an early-onset benign myopathy characterized by proximal muscle weakness and multiple flexion contractures [1–3]. It is caused by dominant mutations in COL6A1 (OMIM # 120220), COL6A2 (OMIM # 120240) [4], and COL6A3 (OMIM # 120250) [5] genes.

Ullrich disease is a form of congenital muscular dystrophy characterized clinically by generalized muscle weakness, contractures of the proximal joints, and hyperflexibility of the distal joints from birth or early infancy. Recently, mutations of the collagen VI gene have been associated with Ullrich disease. The authors report on a boy with Ullrich disease who has complete deficiency of collagen VI and harbors compound heterozygous mutations in the collagen VI alpha 2 gene. Absence of microfibrils on EM, together with normal collagen fibrils and basal lamina, suggests that loss of a link between interstitium and basal lamina may be a new molecular pathomechanism of muscular dystrophy.

X-linked myopathy with excessive autophagy (XMEA) is a childhood-onset disease characterized by progressive vacuolation and atrophy of skeletal muscle. We show that XMEA is caused by hypomorphic alleles of the VMA21 gene, that VMA21 is the diverged human ortholog of the yeast Vma21p protein, and that like Vma21p it is an essential assembly chaperone of the V-ATPase, the principal mammalian proton pump complex. Decreased VMA21 raises lysosomal pH, which reduces lysosomal degradative ability and blocks autophagy. This reduces cellular free amino acids, which upregulates the mTOR pathway and mTOR-dependent macroautophagy, resulting in proliferation of large and ineffective autolysosomes that engulf sections of cytoplasm, merge together, and vacuolate the cell. Our results uncover macroautophagic overcompensation leading to cell vacuolation and tissue atrophy as a mechanism of disease.

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.

Calcium-independent group VIA phospholipase A(2) (iPLA(2)beta), encoded by PLA2G6, has been shown to be involved in various physiological and pathological processes, including immunity, cell death, and cell membrane homeostasis. Mutations in the PLA2G6 gene have been recently identified in patients with infantile neuroaxonal dystrophy (INAD). Subsequently, it was reported that similar neurological impairment occurs in gene-targeted mice with a null mutation of iPLA(2)beta, whose disease onset became apparent approximately 1 to 2 years after birth. Here, we report the establishment of an improved mouse model for INAD that bears a point mutation in the ankyrin repeat domain of Pla2g6 generated by N-ethyl-N-nitrosourea mutagenesis. These mutant mice developed severe motor dysfunction, including abnormal gait and poor performance in the hanging grip test, as early as 7 to 8 weeks of age, in a manner following Mendelian law. Neuropathological examination revealed widespread formation of spheroids containing tubulovesicular membranes similar to human INAD. Molecular and biochemical analysis revealed that the mutant mice expressed Pla2g6 mRNA and protein, but the mutated Pla2g6 protein had no glycerophospholipid-catalyzing enzyme activity. Because of the significantly early onset of the disease, this mouse mutant (Pla2g6-inad) could be highly useful for further studies of pathogenesis and experimental interventions in INAD and neurodegeneration.

FOXO1 (forkhead box O1), a forkhead-type transcription factor whose gene expression is up-regulated in the skeletal muscle during starvation, appears to be a key molecule of energy metabolism and skeletal muscle atrophy. Cathepsin L, a lysosomal proteinase whose expression is also up-regulated in the skeletal muscle during starvation, is induced in transgenic mice overexpressing FOXO1 relative to wild-type littermates. In the present study, we conducted in vivo and in vitro experiments focusing on FOXO1 regulation of Ctsl (cathepsin L gene; CTSL1 in humans) expression in the skeletal muscle. During fasting and refeeding of C57BL/6 mice, Ctsl was regulated in parallel with FOXO1 in the skeletal muscle. Fasting-induced Ctsl expression was attenuated in transgenic mice overexpressing a dominant-negative form of FOXO1 or in skeletal-muscle-specific Foxo1-knockout mice relative to respective wild-type controls. Using C2C12 mouse myoblasts overexpressing a constitutively active form of FOXO1, we showed that FOXO1 induces Ctsl expression. Moreover, we found FOXO1-binding sites in both the mouse Ctsl and human CTSL1 promoters. The luciferase reporter analysis revealed that the mouse Ctsl and human CTSL1 promoters are activated by FOXO1, which is abolished by mutations in the consensus FOXO1-binding sites. Gel mobility-shift and chromatin immunoprecipiation assays showed that FOXO1 is recruited and binds to the Ctsl promoter. The present study provides in vivo and in vitro evidence that Ctsl is a direct target of FOXO1 in the skeletal muscle, thereby suggesting a role for the FOXO1/cathepsin L pathway in fasting-induced skeletal muscle metabolic change and atrophy.

Four-and-a-half LIM domain 1 gene (FHL1) has recently been identified as the causative gene for reducing body myopathy (RBM), X-linked scapuloperoneal myopathy (SPM) and X-linked myopathy with postural muscle atrophy (XMPMA). Rigid spine is a common clinical feature of the three diseases. We searched for FHL1 mutations in eighteen patients clinically diagnosed as rigid spine syndrome (RSS). We identified one RSS patient with FHL1 mutation. Reducing bodies were observed in few fibers of the patient's muscle sample. Amount of FHL1 protein was decreased on immunoblotting. In conclusion, FHL1 can be one of the causative genes for RSS.

Summary Major histocompatibility complex (MHC) class II molecules present antigenic peptides derived from engulfed exogenous proteins to CD4 + T cells. Exogenous antigens are processed in mature endosomes and lysosomes where acidic proteases reside and peptide‐binding to class II alleles is favoured. Hence, maintenance of the microenvironment within these organelles is probably central to efficient MHC class II‐mediated antigen presentation. Lysosome‐associated membrane proteins such as LAMP‐2 reside in mature endosomes and lysosomes, yet their role in exogenous antigen presentation pathways remains untested. In this study, human B cells lacking LAMP‐2 were examined for changes in MHC class II‐restricted antigen presentation. MHC class II presentation of exogenous antigen and peptides to CD4 + T cells was impaired in the LAMP‐2‐deficient B cells. Peptide‐binding to MHC class II on LAMP‐2‐deficient B cells was reduced at physiological pH compared with wild‐type cells. However, peptide‐binding and class II‐restricted antigen presentation were restored by incubation of LAMP‐2‐negative B cells at acidic pH, suggesting that efficient loading of exogenous epitopes by MHC class II molecules is dependent upon LAMP‐2 expression in B cells. Interestingly, class II presentation of an epitope derived from an endogenous transmembrane protein was detected using LAMP‐2‐deficient B cells. Consequently, LAMP‐2 may control the repertoire of peptides displayed by MHC class II molecules on B cells and influence the balance between endogenous and exogenous antigen presentation.

Background Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE) myopathy, also called distal myopathy with rimmed vacuoles (DMRV) or hereditary inclusion body myopathy (HIBM), is a rare, progressive autosomal recessive disorder caused by mutations in the GNE gene. Here, we examined the relationship between genotype and clinical phenotype in participants with GNE myopathy. Methods Participants with GNE myopathy were asked to complete a questionnaire regarding medical history and current symptoms. Results A total of 71 participants with genetically confirmed GNE myopathy (27 males and 44 females; mean age, 43.1±13.0 (mean±SD) years) completed the questionnaire. Initial symptoms (e.g., foot drop and lower limb weakness) appeared at a mean age of 24.8±8.3 years. Among the 71 participants, 11 (15.5%) had the ability to walk, with a median time to loss of ambulation of 17.0±2.1 years after disease onset. Participants with a homozygous mutation (p.V572L) in the N-acetylmannosamine kinase domain (KD/KD participants) had an earlier disease onset compared to compound heterozygous participants with mutations in the uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase and N-acetylmannosamine kinase domains (ED/KD participants; 26.3±7.3 vs. 21.2±11.1 years, respectively). KD/KD participants were more frequently non-ambulatory compared to ED/KD participants at the time of survey (80% vs. 50%). Data were verified using medical records available from 17 outpatient participants. Conclusions Homozygous KD/KD participants exhibited a more severe phenotype compared to heterozygous ED/KD participants.