Estimating prevalence for limb-girdle muscular dystrophy based on public sequencing databases.

PURPOSE: Limb-girdle muscular dystrophies (LGMD) are a genetically heterogeneous category of autosomal inherited muscle diseases. Many genes causing LGMD have been identified, and clinical trials are beginning for treatment of some genetic subtypes. However, even with the gene-level mechanisms known, it is still difficult to get a robust and generalizable prevalence estimation for each subtype due to the limited amount of epidemiology data and the low incidence of LGMDs. METHODS: Taking advantage of recently published exome and genome sequencing data from the general population, we used a Bayesian method to develop a robust disease prevalence estimator. RESULTS: This method was applied to nine recessive LGMD subtypes. The estimated disease prevalence calculated by this method was largely comparable with published estimates from epidemiological studies; however, it highlighted instances of possible underdiagnosis for LGMD2B and 2L. CONCLUSION: The increasing size of aggregated population variant databases will allow for robust and reproducible prevalence estimates of recessive disease, which is critical for the strategic design and prioritization of clinical trials.

Effect of Ibuprofen on Skeletal Muscle of Dysferlin-Null Mice.

Ibuprofen, a nonsteroidal anti-inflammatory drug, and nitric oxide (NO) donors have been reported to reduce the severity of muscular dystrophies in mice associated with the absence of dystrophin or α-sarcoglycan, but their effects on mice that are dystrophic due to the absence of dysferlin have not been examined. We have tested ibuprofen, as well as isosorbide dinitrate (ISDN), a NO donor, to learn whether used alone or together they protect dysferlin-null muscle in A/J mice from large strain injury (LSI) induced by a series of high strain lengthening contractions. Mice were maintained on chow containing ibuprofen and ISDN for 4 weeks. They were then subjected to LSI and maintained on the drugs for 3 additional days. We measured loss of torque immediately following injury and at day 3 postinjury, fiber necrosis, and macrophage infiltration at day 3 postinjury, and serum levels of the drugs at the time of euthanasia. Loss of torque immediately after injury was not altered by the drugs. However, the torque on day 3 postinjury significantly decreased as a function of ibuprofen concentration in the serum (range, 0.67-8.2 µg/ml), independent of ISDN. The effects of ISDN on torque loss at day 3 postinjury were not significant. In long-term studies of dysferlinopathic BlAJ mice, lower doses of ibuprofen had no effects on muscle morphology, but reduced treadmill running by 40%. Our results indicate that ibuprofen can have deleterious effects on dysferlin-null muscle and suggest that its use at pharmacological doses should be avoided by individuals with dysferlinopathies.

Mutations in contactin-1, a neural adhesion and neuromuscular junction protein, cause a familial form of lethal congenital myopathy.

We have previously reported a group of patients with congenital onset weakness associated with a deficiency of members of the syntrophin-alpha-dystrobrevin subcomplex and have demonstrated that loss of syntrophin and dystrobrevin from the sarcolemma of skeletal muscle can also be associated with denervation. Here, we have further studied four individuals from a consanguineous Egyptian family with a lethal congenital myopathy inherited in an autosomal-recessive fashion and characterized by a secondary loss of beta2-syntrophin and alpha-dystrobrevin from the muscle sarcolemma, central nervous system involvement, and fetal akinesia. We performed homozygosity mapping and candidate gene analysis and identified a mutation that segregates with disease within CNTN1, the gene encoding for the neural immunoglobulin family adhesion molecule, contactin-1. Contactin-1 transcripts were markedly decreased on gene-expression arrays of muscle from affected family members compared to controls. We demonstrate that contactin-1 is expressed at the neuromuscular junction (NMJ) in mice and man in addition to the previously documented expression in the central and peripheral nervous system. In patients with secondary dystroglycanopathies, we show that contactin-1 is abnormally localized to the sarcolemma instead of exclusively at the NMJ. The cntn1 null mouse presents with ataxia, progressive muscle weakness, and postnatal lethality, similar to the affected members in this family. We propose that loss of contactin-1 from the NMJ impairs communication or adhesion between nerve and muscle resulting in the severe myopathic phenotype. This disorder is part of the continuum in the clinical spectrum of congenital myopathies and congenital myasthenic syndromes.

Systemic Delivery of Dysferlin Overlap Vectors Provides Long-Term Gene Expression and Functional Improvement for Dysferlinopathy.

Dysferlinopathies comprise a family of disorders caused by mutations in the dysferlin (DYSF) gene, leading to a progressive dystrophy characterized by chronic muscle fiber loss, fat replacement, and fibrosis. To correct the underlying histopathology and function, expression of full-length DYSF is required. Dual adeno-associated virus vectors have been developed, defined by a region of homology, to serve as a substrate for reconstitution of the full 6.5 kb dysferlin cDNA. Previous work studied the efficacy of this treatment through intramuscular and regional delivery routes. To maximize clinical efficacy, dysferlin-deficient mice were treated systemically to target all muscles through the vasculature for efficacy and safety studies. Mice were evaluated at multiple time points between 4 and 13 months post treatment for dysferlin expression and functional improvement using magnetic resonance imaging and magnetic resonance spectroscopy and membrane repair. A systemic dose of 6 × 1012 vector genomes resulted in widespread gene expression in the muscles. Treated muscles showed a significant decrease in central nucleation, collagen deposition, and improvement of membrane repair to wild-type levels. Treated gluteus muscles were significantly improved compared to placebo-treated muscles and were equivalent to wild type in volume, intra- and extramyocellular lipid accumulation, and fat percentage using magnetic resonance imaging and magnetic resonance spectroscopy. Dual-vector treatment allows for production of full-length functional dysferlin with no toxicity. This confirms previous safety data and validates translation of systemic gene delivery for dysferlinopathy patients.

Complete deletion of all alpha-dystrobrevin isoforms does not reveal new neuromuscular junction phenotype.

The dystrophin glycoprotein complex (DGC) is critical for muscle stability, and mutations in DGC proteins lead to muscular dystrophy. The DGC also contributes to the maturation and maintenance of the neuromuscular junction (NMJ). The gene encoding the DGC protein alpha-dystrobrevin undergoes alternative splicing to produce at least five known isoforms. Isoform-specific antibody staining and reverse transcription PCR in mutant mice with a deletion of exon 3 of the alpha-dystrobrevin gene suggested the existence of a remaining synaptic isoform, which might be compensating for alpha-dystrobrevin function. To test this possibility and to more completely understand the synaptic function of alpha-dystrobrevin, we used a two-step homologous recombination strategy combined with in vivo Cre-mediated excision to generate mice with a large deletion of the alpha-dystrobrevin gene to disrupt all isoforms. However, these mice did not exhibit a more severe NMJ phenotype than that observed in the exon 3-deleted mice. Nonetheless, these mice not only eliminate possible compensation by remaining isoforms of alpha-dystrobrevin, but also offer a conditional allele that could be used to identify tissue-specific and developmental functions of alpha-dystrobrevin. This work also demonstrates a successful strategy to achieve deletion of a large genomic sequence, which can be a valuable tool for functional studies of genes encoding multiple isoforms that span a large genomic region.

6th Dysferlin Conference, 3-6 April 2013, Arlington, Virginia, USA.

The 2013 Dysferlin Conference, sponsored and organized by the Jain Foundation, was held from April 3-6, 2013 in Arlington, VA. Participants included 34 researcher speakers, 5 dysferlinopathy patients and all 8 members of the Jain Foundation team. Dysferlinopathy is a rare disease that typically robs patients of mobility during their second or third decade of life. The goals of these Dysferlin Conferences are to bring experts in the field together so that they will collaborate with one another, to quicken the pace of understanding the biology of the disease and to build effective platforms to ameliorate disease. This is important because the function of dysferlin and how to compensate for its absence is still not well understood, in spite of the fact that the dysferlin gene was identified more than a decade ago. The objective of this conference, therefore, was to share and discuss the newest unpublished research defining the role of dysferlin in skeletal muscle, why its absence causes muscular dystrophy and possible therapies for dysferlin-deficient muscular dystrophy patients.

The light chains of microtubule-associated proteins MAP1A and MAP1B interact with α1-syntrophin in the central and peripheral nervous system.

Microtubule-associated proteins of the MAP1 family (MAP1A, MAP1B, and MAP1S) share, among other features, a highly conserved COOH-terminal domain approximately 125 amino acids in length. We conducted a yeast 2-hybrid screen to search for proteins interacting with this domain and identified α1-syntrophin, a member of a multigene family of adapter proteins involved in signal transduction. We further demonstrate that the interaction between the conserved COOH-terminal 125-amino acid domain (which is located in the light chains of MAP1A, MAP1B, and MAP1S) and α1-syntrophin is direct and occurs through the pleckstrin homology domain 2 (PH2) and the postsynaptic density protein 95/disk large/zonula occludens-1 protein homology domain (PDZ) of α1-syntrophin. We confirmed the interaction of MAP1B and α1-syntrophin by co-localization of the two proteins in transfected cells and by co-immunoprecipitation experiments from mouse brain. In addition, we show that MAP1B and α1-syntrophin partially co-localize in Schwann cells of the murine sciatic nerve during postnatal development and in the adult. However, intracellular localization of α1-syntrophin and other Schwann cell proteins such as ezrin and dystrophin-related protein 2 (DRP2) and the localization of the axonal node of Ranvier-associated protein Caspr1/paranodin were not affected in MAP1B null mice. Our findings add to a growing body of evidence that classical MAPs are likely to be involved in signal transduction not only by directly modulating microtubule function, but also through their interaction with signal transduction proteins.

The ABCA1 cholesterol transporter associates with one of two distinct dystrophin-based scaffolds in Schwann cells.

Cytoskeletal scaffolding complexes help organize specialized membrane domains with unique functions on the surface of cells. In this study, we define the scaffolding potential of the Schwann cell dystrophin glycoprotein complex (DGC) by establishing the presence of four syntrophin isoforms, (alpha1, beta1, beta2, and gamma2), and one dystrobrevin isoform, (alpha-dystrobrevin-1), in the abaxonal membrane. Furthermore, we demonstrate the existence of two separate DGCs in Schwann cells that divide the abaxonal membrane into spatially distinct domains, the DRP2/periaxin rich plaques and the Cajal bands that contain Dp116, utrophin, alpha-dystrobrevin-1 and four syntrophin isoforms. Finally, we show that the two different DGCs can scaffold unique accessory molecules in distinct areas of the Schwann cell membrane. Specifically, the cholesterol transporter ABCA1, associates with the Dp116/syntrophin complex in Cajal bands and is excluded from the DRP2/periaxin rich plaques.

Differential targeting of nNOS and AQP4 to dystrophin-deficient sarcolemma by membrane-directed alpha-dystrobrevin.

alpha-Dystrobrevin associates with and is a homologue of dystrophin, the protein linked to Duchenne and Becker muscular dystrophies. We used a transgenic approach to restore alpha-dystrobrevin to the sarcolemma in mice that lack dystrophin (mdx mice) to study two interrelated functions: (1) the ability of alpha-dystrobrevin to rescue components of the dystrophin complex in the absence of dystrophin and (2) the ability of sarcolemmal alpha-dystrobrevin to ameliorate the dystrophic phenotype. We generated transgenic mice expressing alpha-dystrobrevin-2a linked to a palmitoylation signal sequence and bred them onto the alpha-dystrobrevin-null and mdx backgrounds. Expression of palmitoylated alpha-dystrobrevin prevented the muscular dystrophy observed in the alpha-dystrobrevin-null mice, demonstrating that the altered form of alpha-dystrobrevin was functional. On the mdx background, the palmitoylated form of alpha-dystrobrevin was expressed on the sarcolemma but did not significantly ameliorate the muscular dystrophy phenotype. Palmitoylated dystrobrevin restored alpha-syntrophin and aquaporin-4 (AQP4) to the mdx sarcolemma but was unable to recruit beta-dystroglycan or the sarcoglycans. Despite restoration of sarcolemmal alpha-syntrophin, neuronal nitric oxide synthase (nNOS) was not localized to the sarcolemma, suggesting that nNOS requires both dystrophin and alpha-syntrophin for correct localization. Thus, although nNOS and AQP4 both require interaction with the PDZ domain of alpha-syntrophin for sarcolemmal association, their localization is regulated differentially.

gamma-Syntrophin scaffolding is spatially and functionally distinct from that of the alpha/beta syntrophins.

The syntrophins are a family of scaffolding proteins with multiple protein interaction domains that link signaling proteins to dystrophin family members. Each of the three most characterized syntrophins (alpha, beta1, beta2) contains a PDZ domain that binds a unique set of signaling proteins including kinases, ion and water channels, and neuronal nitric oxide synthase (nNOS). The PDZ domains of the gamma-syntrophins do not bind nNOS. In vitro pull-down assays show that the gamma-syntrophins can bind dystrophin but have unique preferences for the syntrophin binding sites of dystrophin family members. Despite their ability to bind dystrophin in vitro, neither gamma-syntrophin isoform co-localizes with dystrophin in skeletal muscle. Furthermore, gamma-syntrophins do not co-purify with dystrophin isolated from mouse tissue. These data suggest that the interaction of gamma-syntrophin with dystrophin is transient and potentially subject to regulatory mechanisms. gamma1-Syntrophin is highly expressed in brain and is specifically localized in hippocampal pyramidal neurons, Purkinje neurons in cerebellum, and cortical neurons. gamma2-Syntrophin is expressed in many tissues including skeletal muscle where it is found only in the subsynaptic space beneath the neuromuscular junction. In both neurons and muscle, gamma-syntrophin isoforms localize to the endoplasmic reticulum where they may form a scaffold for signaling and trafficking.

Syntrophins and dystrobrevins: defining the dystrophin scaffold at synapses.

Dystrophin and its associated proteins were originally identified in skeletal muscle, where the complex provides mechanical stabilization to the sarcolemma during contraction. However, the dystrophin complex is also present at membrane specializations in many non-muscle cells, including synaptic sites in neurons. The function of the dystrophin complex at these sites is still unknown, but emerging results suggest that the dystrophin complex can function as a scaffold for signaling proteins. In this review, we examine the growing body of evidence that suggests the dystrophin complex may have a dual function: membrane stabilization and transmembrane signaling. We focus on the role of two dystrophin-associated proteins, syntrophin and dystrobrevin, in the formation of a signaling scaffold and review evidence suggesting a role in synapse formation and maintenance.

DAMAGE, a novel alpha-dystrobrevin-associated MAGE protein in dystrophin complexes.

Mice rendered null for alpha-dystrobrevin, a component of the dystrophin complex, have muscular dystrophy, despite the fact that the sarcolemma remains relatively intact (Grady, R. M., Grange, R. W., Lau, K. S., Maimone, M. M., Nichol, M. C., Stull, J. T., and Sanes, J. R. (1999) Nat. Cell Biol. 1, 215-220) Thus, alpha-dystrobrevin may serve a signaling function that is important for the maintenance of muscle integrity. We have identified a new dystrobrevin-associated protein, DAMAGE, that may play a signaling role in brain, muscle, and peripheral nerve. In humans, DAMAGE is encoded by an intronless gene located at chromosome Xq13.1, a locus that contains genes involved in mental retardation. DAMAGE associates directly with alpha-dystrobrevin, as shown by yeast two-hybrid, and co-immunoprecipitates with the dystrobrevin-syntrophin complex from brain. This co-immunoprecipitation is dependent on the presence of alpha-dystrobrevin but not beta-dystrobrevin. The DAMAGE protein contains a potential nuclear localization signal, 30 12-amino acid repeats, and two MAGE homology domains. The domain structure of DAMAGE is similar to that of NRAGE, a MAGE protein that mediates p75 neurotrophin receptor signaling and neuronal apoptosis (Salehi, A. H., Roux, P. P., Kubu, C. J., Zeindler, C., Bhakar, A., Tannis, L. L., Verdi, J. M., and Barker, P. A. (2000) Neuron 27, 279-288). DAMAGE is highly expressed in brain and is present in the cell bodies and dendrites of hippocampal and Purkinje neurons. In skeletal muscle, DAMAGE is at the postsynaptic membrane and is associated with a subset of myonuclei. DAMAGE is also expressed in peripheral nerve, where it localizes along with other members of the dystrophin complex to the perineurium and myelin. These results expand the role of dystrobrevin and the dystrophin complex in membrane signaling and disease.