Dystrophin plays an important role in muscle contraction linking the intracellular cytoskeleton to the extracellular matrix. Mutations of the dystrophin gene leading to a complete loss of the protein cause Duchenne muscular dystrophy (DMD), frequently associated with severe cardiomyopathy. Early clinical trials in DMD using gene transfer to skeletal muscle are underway, but gene transfer to dystrophic cardiac muscle has not been tested in humans yet. The aim of this study was to develop an optimised protocol for cardiac gene therapy in the mouse model of dystrophin-deficiency (mdx) using a cardiac promoter for expression of a microdystrophin (µDys) transgene packaged into an adeno-associated virus (AAV) 9 vector. In this study adult mdx mice were intravenously injected with 1 x 1012 genomic particles of AAV9 vectors containing a cDNA encoding µDys under control of either an ubiquitously active CMV promoter (CMV) or a cardiac specific CMV-enhanced myosin light chain (MLC0.26) promoter. Mice were challenged by voluntary wheel exercise and analysed over 10 months following gene transfer. Both AAV9 vectors led to sustained µDys expression in cardiac muscle, but the MLC promoter conferred about 4-fold higher protein levels. AAV9-CMV-MLC0.26-µDys resulted in a significant protection of cardiac morphology and function as assessed by histopathology, serial echocardiograms and left ventricular catheterization/pressure volume loop measurements. The authors of this study have established an AAV9-mediated gene transfer approach for efficient and specific long-term µDys-expression in the hearts of mdx mice, resulting in a sustained therapeutic effect. Thus, this approach might be a basis for further translation into a treatment strategy for DMD-associated cardiomyopathy.

Long-term preservation of cardiac structure and function after AAV9-mediated microdystrophin gene transfer in mdx mice. Schinkel S, Bauer R, Bekeredjian R, Stucka R, Rutschow D, Lochmüller H, Kleinschmidt JA, Katus HA, Müller OJ. Hum Gene Ther. 2012 Jan 16. [Epub ahead of print]

Increased utrophin expression is known to reduce pathology in dystrophin-deficient skeletal muscles. Transgenic over-expression of PGC-1á has been shown to increase levels of utrophin mRNA and improve the histology of mdx muscles. Other reports have shown that PGC-1á signaling can lead to increased oxidative capacity and a fast to slow fiber type shift. Given that it has been shown that slow fibers produce and maintain more utrophin than fast skeletal muscle fibers, the authors hypothesized that over-expression of PGC-1á in post-natal mdx mice would increase utrophin levels via a fiber type shift, resulting in more slow, oxidative fibers that are also more resistant to contraction-induced damage. To test this hypothesis, neonatal mdx mice were injected with recombinant adeno-associated virus (AAV) driving expression of PGC-1á. PGC-1á over-expression resulted in increased utrophin and type I myosin heavy chain expression as well as elevated mitochondrial protein expression. Muscles were shown to be more resistant to contraction-induced damage and more fatigue resistant. Sirt-1 was increased while p38 activation and NRF-1 were reduced in PGC-1á over-expressing muscle when compared to control. Whether the use of a pharmacological PGC-1á pathway activator, resveratrol, could drive the same physiological changes was also evaluated. Resveratrol administration (100 mg/kg/day) resulted in improved fatigue resistance, but did not achieve significant increases in utrophin expression. These data suggest that the PGC-1á pathway is a potential target for therapeutic intervention in dystrophic skeletal muscle.

Rescue of Dystrophic Skeletal Muscle by PGC-1á Involves a Fast to Slow Fiber Type Shift in the mdx Mouse. Selsby JT, Morine KJ, Pendrak K, Barton ER, Sweeney HL. PLoS One. 2012;7(1):e30063. Epub 2012 Jan 11. 

γ-Sarcoglycanopathy or limb girdle muscular dystrophy type 2C is an untreatable disease caused by autosomal recessively inherited mutations of the γ-sarcoglycan gene. Nine non-ambulatory patients (two males, seven females, mean age 27 years; range 16-38 years) with del525T homozygous mutation of the γ-sarcoglycan gene and no γ-sarcoglycan immunostaining on muscle biopsy were divided into three equal groups to receive three escalating doses of an adeno-associated virus serotype 1 vector expressing the human γ-sarcoglycan gene under the control of the desmin promoter, by local injection into the extensor carpi radialis muscle. The first group received a single injection of 3 × 10(9) viral genomes in 100 µl, the second group received a single injection of 1.5 × 10(10) viral genomes in 100 µl, and the third group received three simultaneous 100-µl injections at the same site, delivering a total dose of 4.5 × 10(10) viral genomes. No serious adverse effects occurred during 6 months of follow-up. All nine patients became adeno-associated virus serotype 1 seropositive and one developed a cytotoxic response to the adeno-associated virus serotype 1 capsid. Thirty days later, immunohistochemical analysis of injected-muscle biopsy specimens showed γ-sarcoglycan expression in all three patients who received the highest dose (4.7-10.5% positively stained fibres), while real-time polymerase chain reaction detected γ-sarcoglycan messenger RNA. In one patient, γ-sarcoglycan protein was detected by western blot. For two other patients who received the low and intermediate doses, discrete levels of γ-sarcoglycan expression (<1% positively stained fibres) were also detectable. Expression of γ-sarcoglycan protein can be induced in patients with limb girdle muscular dystrophy type 2C by adeno-associated virus serotype 1 gene transfer, with no serious adverse effects.

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Limb girdle muscular dystrophy 1D/1E (OMIM nomenclature LGMD1D, Human Gene Nomenclature Committee LGMD1E), a skeletal and cardiac myopathy, has previously been linked to chromosome 6q23. Herein the authors used laser capture microdissection to isolate cytoplasmic inclusions from skeletal muscle from a patient with LGMD1D/1E, performed mass spectrometry-based proteomics on these minute inclusions, and identified through bioinformatics desmin as their major constituent. Sequencing in this patient and family members identified the genetic basis of the previously reported 6q23 linked LGMD1D/1E to be due to an intron splice donor site mutation (IVS3+3A>G) of the desmin gene located on chromosome 2q35.

Etiology of limb girdle muscular dystrophy 1D/1E determined by laser capture microdissection proteomics. Greenberg SA, Salajegheh M, Judge DP, Feldman MW, Kuncl RW, Waldon Z, Steen H, Wagner KR. Ann Neurol. 2012 Jan;71(1):141-5. doi: 10.1002/ana.22649.

This study explored the possibility of copy number mutations in patients with Charcot-Marie-Tooth disease (CMT) whose responsible genes remain undefined, on the basis of the hypothesis that copy number mutations of the genes encoding myelin compact proteins are responsible for myelin disorders in humans. A family with 6 affected members in 3 consecutive generations, presenting with motor and sensory demyelinating polyneuropathy, was investigated. Characteristic clinical features in this pedigree include Adie pupils and substantial intrafamilial variability in the age at onset, electrophysiological findings, and clinical severity. Nucleotide sequence analyses of PMP22, MPZ, or GJB1 and gene dosage study of PMP22 did not reveal causative mutations. Hence, the authors applied a custom-designed array for comparative genomic hybridization (CGH) analysis to conduct a comprehensive screening of copy number mutations involving any of the known causative genes for CMT other than PMP22. The array CGH analyses revealed increased gene dosage involving the whole MPZ, and the flanking genes of SDHC and C1orf192. The gene dosage is estimated to be 5 copies. This mutation showed complete cosegregation with the disease phenotype in this pedigree. The increased gene dosage of MPZ and increased expression level of MPZ mRNA emphasise the important role of the dosage of the MPZ protein in the functional integrity of peripheral nerve myelin in humans, and provide a new insight into the pathogenic mechanisms underlying CMT.

Researchers have now identified the genes and proteins, which damage muscle cells, as well as the mechanisms that can cause a common form of muscular dystrophy. The discovery made by an international team of researchers led by a scientist at Fred Hutchinson Cancer Research Center could lead to a biomarker-based test for diagnosing facioscapulohumeral muscular dystrophy (FSHD), and the findings have implications for developing future treatments as well as for cancer immunotherapies in general. The work established a viable roadmap for how the expression of the DUX4 gene can cause FSHD. Whether this is the sole cause of FSHD is not known; however, the latest findings are strong evidence of the genetic link. Dr. Tapscott and colleagues sought answers to the questions about what the DUX4 protein does both normally in the body and in the FSHD disease process. In the latest study, they identified that the DUX4 protein regulates many genes that are normally expressed in the male germ line but are abnormally expressed in FSHD muscle. This study is a significant step forward by solidifying that the DUX4 transcription factor causes this disease, while offering a number of viable mechanisms for why the muscle is damaged. Now that scientists know that targets for DUX4 are expressed in skeletal muscle, an antibody- or RNA-based test could be developed to diagnose FSHD by examining muscle tissue from a biopsy. Such biomarker-based tests also could be used to determine how well new treatments are working to suppress FSHD.

DUX4 Activates Germline Genes, Retroelements, and Immune Mediators: Implications for Facioscapulohumeral Dystrophy. Geng LN, Yao Z, Snider L, Fong AP, Cech JN, Young JM, van der Maarel SM, Ruzzo WL, Gentleman RC, Tawil R, Tapscott SJ. Dev Cell. 2011 Dec 28.

The coexistence of systemic lupus erythematosus (SLE) and myasthenia gravis (MG) is rarely reported, and most of the published studies are case reports. Hydroxychloroquine, an antimalarial agent, is an essential treatment in patients with SLE but special caution is recommended when used in MG patients. Herein, the authors retrospectively analyzed the clinical features, laboratory findings, and outcome of 17 patients with both diseases with a special focus regarding hydroxychloroquine use and with a review of the literature. All patients were women. The mean age at MG onset and SLE diagnosis was 34.5 [14-64] and 37.8 [18-72] years, respectively. The presenting symptoms of MG were limb weakness (94%), ocular (88%) and bulbar involvement (53%). Autoantibodies against the acetylcholine receptor were positive in 94% of cases. The main manifestations of SLE included arthritis (88%), cytopenias (53%) and skin rash (41%). Treatment of SLE required hydroxychloroquine (94%), steroids (47%) and immunosuppressive drugs (18%). Among eight patients (47%) who developed MG after initiation of hydroxychloroquine, the question of induction of MG by hydroxychloroquine was raised in one patient. On the other hand, an exacerbation of myasthenic symptoms was only seen in one of the eight patients who received hydroxychloroquine after the diagnosis of MG. Including the cases in this study, a total of 70 patients with SLE and MG were reviewed. Compared with a large series of 1,000 unselected SLE patients, those with associated MG were older, had lower incidence of cutaneous, renal, and neurological manifestations, and higher frequency of anticardiolipin antibodies and lupus anticoagulant. In conclusion, the clinical pattern of patients with SLE and MG seems to be characterized by a less severe course of SLE and higher frequency of antiphospholipid antibodies. Hydroxychloroquine treatment appears to be safe in this setting.

The association of systemic lupus erythematosus and myasthenia gravis: a series of 17 cases, with a special focus on hydroxychloroquine use and a review of the literature. Jallouli M, Saadoun D, Eymard B, Leroux G, Haroche J, Le Thi Huong D, De Gennes C, Chapelon C, Benveniste O, Wechsler B, Cacoub P, Amoura Z, Piette JC, Costedoat-Chalumeau N. J Neurol. 2011 Dec 8. [Epub ahead of print]

The precise function of an enzyme critical for normal muscle structure which is also involved in several muscular dystrophies has been identified by researchers at the University of Iowa. The findings could be used to develop rapid, large-scale testing of potential muscular dystrophy therapies. The enzyme, called LARGE, adds a critical sugar chain onto dystroglycan, thus reinforcing cell membranes in many tissues including muscle and brain. Dystroglycan does not function properly without this sugar link, resulting in muscular dystrophies and brain abnormalities. The new study shows that the enzyme activity of LARGE transfers the sugars xylose and glucuronic acid. Using nuclear magnetic resonance analysis (NMR), the team was also able to determine the precise structure of the sugar chain produced by LARGE, which has not previously been observed. The study confirmed that this unique sugar chain is responsible for dystroglycan's ability to link proteins such as laminin in muscle and neurexin in brain. In addition to LARGE, several other enzymes are involved in building the important dystroglycan sugar chain, and mutations in all these enzymes cause congenital muscular dystrophies collectively known as secondary dystroglycanopathies. These disorders include Fukuyama Congenital Muscular Dystrophy, Walker-Warburg Syndrome, Muscle-Eye-Brain disease, Congenital Muscular Dystrophy 1C and 1D, and limb-girdle muscular dystrophy 2I. However, in all cases, the part of the sugar chain that is critical for dystroglycan function is the part that is added by LARGE. By understanding the function of the LARGE enzyme, the researchers have now been able to develop an enzyme assay, which could be used in a large-scale high-throughput screen for drugs that increase (or decrease) LARGE activity. Using the assay to identify compounds that boost LARGE activity might lead to potential treatments for the secondary dystroglycanopathies. The assay could also be used to look at variations in LARGE activity in patients' cells. This may help identify patients who are affected by these LARGE-related muscular dystrophies.

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> Dystroglycan function requires xylosyl- and glucuronyltransferase activities of LARGE. Inamori K, Yoshida-Moriguchi T, Hara Y, Anderson ME, Yu L, Campbell KP. Science. 2012 Jan 6;335(6064):93-6.

Little is known about the vascular function and expression of endothelial and neuronal nitric oxide synthases (eNOS and nNOS) in Duchenne muscular dystrophy (DMD). Bradykinin is involved in the regulation of eNOS expression induced by angiotensin-converting enzyme inhibitors. We characterized the vascular function and eNOS and nNOS expression in a canine model of DMD and evaluated the effects of chronic bradykinin treatment. Vascular function was examined in conscious golden retriever muscular dystrophy (GRMD) dogs with left ventricular dysfunction (measured by echocardiography) and in isolated coronary arteries. eNOS and nNOS proteins in carotid arteries were measured by western blot and cyclic guanosine monophosphate (cGMP) content was analyzed by radioimmunoassay. Compared with controls, GRMD dogs had an impaired vasodilator response to acetylcholine. In isolated coronary artery, acetylcholine-elicited relaxation was nearly absent in placebo-treated GRMD dogs. This was explained by reduced nNOS and eNOS proteins and cGMP content in arterial tissues. Chronic bradykinin infusion (1 ìg/min, 4 weeks) restored in vivo and in vitro vascular response to acetylcholine to the level of control dogs. This effect was NO-mediated through upregulation of eNOS and nNOS expression. In conclusion, this study is the first to demonstrate that DMD is associated with NO-mediated vascular endothelial dysfunction linked to an altered expression of eNOS and nNOS, which can be overcome by bradykinin.

Vascular endothelial dysfunction in Duchenne muscular dystrophy is restored by bradykinin through upregulation of eNOS and nNOS. Dabiré H, Barthélémy I, Blanchard-Gutton N, Sambin L, Sampedrano CC, Gouni V, Unterfinger Y, Aguilar P, Thibaud JL, Ghaleh B, Bizé A, Pouchelon JL, Blot S, Berdeaux A, Hittinger L, Chetboul V, Su JB. Basic Res Cardiol. 2012 Jan;107(1):1-9.

Charcot-Marie-Tooth neuropathy has been reported to be associated with renal diseases, mostly focal segmental glomerulosclerosis (FSGS). However, the common mechanisms underlying the neuropathy and FSGS remain unknown. Mutations in INF2 were recently identified in patients with autosomal dominant FSGS. INF2 encodes a formin protein that interacts with the Rho-GTPase CDC42 and myelin and lymphocyte protein (MAL) that are implicated in essential steps of myelination and myelin maintenance. The authors of this study therefore hypothesized that INF2 may be responsible for cases of Charcot-Marie-Tooth neuropathy associated with FSGS. They performed direct genotyping of INF2 in 16 index patients with Charcot-Marie-Tooth neuropathy and FSGS who did not have a mutation in PMP22 or MPZ, encoding peripheral myelin protein 22 and myelin protein zero, respectively. Histologic and functional studies were also conducted. Nine new heterozygous mutations in 12 of the 16 index patients (75%) were identified, all located in exons 2 and 3, encoding the diaphanous-inhibitory domain of INF2. Patients presented with an intermediate form of Charcot-Marie-Tooth neuropathy as well as a glomerulopathy with FSGS on kidney biopsy. Immunohistochemical analysis revealed strong INF2 expression in Schwann-cell cytoplasm and podocytes. Moreover, it was demonstrated that INF2 colocalizes and interacts with MAL in Schwann cells. The INF2 mutants perturbed the INF2-MAL-CDC42 pathway, resulting in cytoskeleton disorganization, enhanced INF2 binding to CDC42 and mislocalization of INF2, MAL, and CDC42. INF2 mutations appear to cause many cases of FSGS-associated Charcot-Marie-Tooth neuropathy, showing that INF2 is involved in a disease affecting both the kidney glomerulus and the peripheral nervous system. These findings provide new insights into the pathophysiological mechanisms linking formin proteins to podocyte and Schwann-cell function. (Funded by the Agence Nationale de la Recherche and others.).

INF2 mutations in Charcot-Marie-Tooth disease with glomerulopathy. Boyer O, Nevo F, Plaisier E, Funalot B, Gribouval O, Benoit G, Cong EH, Arrondel C, Tête MJ, Montjean R, Richard L, Karras A, Pouteil-Noble C, Balafrej L, Bonnardeaux A, Canaud G, Charasse C, Dantal J, Deschenes G, Deteix P, Dubourg O, Petiot P, Pouthier D, Leguern E, Guiochon-Mantel A, Broutin I, Gubler MC, Saunier S, Ronco P, Vallat JM, Alonso MA, Antignac C, Mollet G. N Engl J Med. 2011 Dec 22;365(25):2377-88.