Phenotypic Spectrum of α-Dystroglycanopathies Associated With the c.919T>a Variant in the FKRP Gene in Humans and Mice.

Mutations in the fukutin-related protein gene, FKRP, are the most frequent single cause of α-dystroglycanopathy. Rare FKRP mutations are clinically not well characterized. Here, we review the phenotype associated with the rare c.919T>A mutation in FKRP in humans and mice. We describe clinical and paraclinical findings in 6 patients, 2 homozygous, and 4-compound heterozygous for c.919T>A, and compare findings with a mouse model we generated, which is homozygous for the same mutation. In patients, the mutation at the homozygous state is associated with a severe congenital muscular dystrophy phenotype invariably characterized by severe multisystem disease and early death. Compound heterozygous patients have a severe limb-girdle muscular dystrophy phenotype, loss of ambulation before age 20 and respiratory insufficiency. In contrast, mice homozygous for the same mutation show no symptoms or signs of muscle disease. Evidence therefore defines the FKRP c.919T>A as a very severe mutation in humans. The huge discrepancy between phenotypes in humans and mice suggests that differences in protein folding/processing exist between human and mouse Fkrp. This emphasizes the need for more detailed structural analyses of FKRP and shows the challenges of developing appropriate animal models of dystroglycanopathies that mimic the disease course in humans.

Degree of Cajal-Retzius Cell Mislocalization Correlates with the Severity of Structural Brain Defects in Mouse Models of Dystroglycanopathy.

The secondary dystroglycanopathies are characterized by the hypoglycosylation of alpha dystroglycan, and are associated with mutations in at least 18 genes that act on the glycosylation of this cell surface receptor rather than the Dag1 gene itself. At the severe end of the disease spectrum, there are substantial structural brain defects, the most striking of which is often cobblestone lissencephaly. The aim of this study was to determine the gene-specific aspects of the dystroglycanopathy brain phenotype through a detailed investigation of the structural brain defects present at birth in three mouse models of dystroglycanopathy-the FKRP(KD) , which has an 80% reduction in Fkrp transcript levels; the Pomgnt1null , which carries a deletion of exons 7-16 of the Pomgnt1 gene; and the Large(myd) mouse, which carries a deletion of exons 5-7 of the Large gene. We show a rostrocaudal and mediolateral gradient in the severity of brain lesions in FKRP(KD) , and to a lesser extent Pomgnt1null mice. Furthermore, the mislocalization of Cajal-Retzius cells is correlated with the gradient of these lesions and the severity of the brain phenotype in these models. Overall these observations implicate gene-specific differences in the pathogenesis of brain lesions in this group of disorders.

A new monoclonal antibody DAG-6F4 against human alpha-dystroglycan reveals reduced core protein in some, but not all, dystroglycanopathy patients.

We generated a novel monoclonal antibody, DAG-6F4, against alpha-dystroglycan which immunolabels the sarcolemma in human muscle biopsies. Its seven amino-acid epitope, PNQRPEL, was identified using phage-displayed peptides and is located immediately after the highly-glycosylated mucin domain of alpha-dystroglycan. On Western blots of recombinant alpha-dystroglycan, epitope accessibility was reduced, but not entirely prevented, by glycosylation. DAG-6F4 immunolabelling was markedly reduced in muscle biopsies from Duchenne muscular dystrophy patients consistent with disruption of the dystroglycan complex. In a range of dystroglycanopathy patients with reduced/altered glycosylation, staining by DAG-6F4 was often less reduced than staining by IIH6 (antibody against the glycan epitope added by LARGE and commonly used to identify glycosylated alpha-dystroglycan). Whereas IIH6 was reduced in all patients, DAG-6F4 was hardly changed in a LARGE patient, less reduced than IIH6 in limb-girdle muscular dystrophy type 2I, but as reduced as IIH6 in some congenital muscular dystrophy patients. Although absence of the LARGE-dependent laminin-binding site appears not to affect alpha-dystroglycan stability at the sarcolemma, the results suggest that further reduction in aDG glycosylation may reduce its stability. These studies suggest that DAG-6F4 may be a useful addition to the antibody repertoire for evaluating the dystroglycan complex in neuromuscular disorders.

Calcium homeostasis in myogenic differentiation factor 1 (MyoD)-transformed, virally-transduced, skin-derived equine myotubes.

Dysfunctional skeletal muscle calcium homeostasis plays a central role in the pathophysiology of several human and animal skeletal muscle disorders, in particular, genetic disorders associated with ryanodine receptor 1 (RYR1) mutations, such as malignant hyperthermia, central core disease, multiminicore disease and certain centronuclear myopathies. In addition, aberrant skeletal muscle calcium handling is believed to play a pivotal role in the highly prevalent disorder of Thoroughbred racehorses, known as Recurrent Exertional Rhabdomyolysis. Traditionally, such defects were studied in human and equine subjects by examining the contractile responses of biopsied muscle strips exposed to caffeine, a potent RYR1 agonist. However, this test is not widely available and, due to its invasive nature, is potentially less suitable for valuable animals in training or in the human paediatric setting. Furthermore, increasingly, RYR1 gene polymorphisms (of unknown pathogenicity and significance) are being identified through next generation sequencing projects. Consequently, we have investigated a less invasive test that can be used to study calcium homeostasis in cultured, skin-derived fibroblasts that are converted to the muscle lineage by viral transduction with a MyoD (myogenic differentiation 1) transgene. Similar models have been utilised to examine calcium homeostasis in human patient cells, however, to date, there has been no detailed assessment of the cells' calcium homeostasis, and in particular, the responses to agonists and antagonists of RYR1. Here we describe experiments conducted to assess calcium handling of the cells and examine responses to treatment with dantrolene, a drug commonly used for prophylaxis of recurrent exertional rhabdomyolysis in horses and malignant hyperthermia in humans.

The transgenic expression of LARGE exacerbates the muscle phenotype of dystroglycanopathy mice.

Mutations in fukutin-related protein (FKRP) underlie a group of muscular dystrophies associated with the hypoglycosylation of α-dystroglycan (α-DG), a proportion of which show central nervous system involvement. Our original FKRP knock-down mouse (FKRP(KD)) replicated many of the characteristics seen in patients at the severe end of the dystroglycanopathy spectrum but died perinatally precluding its full phenotyping and use in testing potential therapies. We have now overcome this by crossing FKRP(KD) mice with those expressing Cre recombinase under the Sox1 promoter. Owing to our original targeting strategy, this has resulted in the restoration of Fkrp levels in the central nervous system but not the muscle, thereby generating a new model (FKRP(MD)) which develops a progressive muscular dystrophy resembling what is observed in limb girdle muscular dystrophy. Like-acetylglucosaminyltransferase (LARGE) is a bifunctional glycosyltransferase previously shown to hyperglycosylate α-DG. To investigate the therapeutic potential of LARGE up-regulation, we have now crossed the FKRP(MD) line with one overexpressing LARGE and show that, contrary to expectation, this results in a worsening of the muscle pathology implying that any future strategies based upon LARGE up-regulation require careful management.

Adenovirus-mediated expression of myogenic differentiation factor 1 (MyoD) in equine and human dermal fibroblasts enables their conversion to caffeine-sensitive myotubes.

Several human and animal myopathies, such as malignant hyperthermia (MH), central core disease and equine recurrent exertional rhabdomyolysis (RER) are confirmed or thought to be associated with dysfunction of skeletal muscle calcium regulation. For some patients in whom the genetic cause is unknown, or when mutational analysis reveals genetic variants with unclear pathogenicity, defects are further studied through use of muscle histopathology and in vitro contraction tests, the latter in particular, when assessing responses to ryanodine receptor agonists, such as caffeine. However, since muscle biopsy is not always suitable, researchers have used cultured cells to model these diseases, by examining calcium regulation in myotubes derived from skin, following forced expression of muscle-specific transcription factors. Here we describe a novel adenoviral vector that we used to express equine MyoD in dermal fibroblasts. In permissive conditions, transduced equine and human fibroblasts differentiated into multinucleated myotubes. We demonstrate that these cells have a functional excitation-calcium release mechanism and, similarly to primary muscle-derived myotubes, respond in a dose-dependent manner to increasing concentrations of caffeine. MyoD-induced conversion of equine skin-derived fibroblasts offers an attractive method for evaluating calcium homeostasis defects in vitro without the need for invasive muscle biopsy.

Defects in glycosylation impair satellite stem cell function and niche composition in the muscles of the dystrophic Large(myd) mouse.

The dystrophin-associated glycoprotein complex (DGC) is found at the muscle fiber sarcolemma and forms an essential structural link between the basal lamina and internal cytoskeleton. In a set of muscular dystrophies known as the dystroglycanopathies, hypoglycosylation of the DGC component α-dystroglycan results in reduced binding to basal lamina components, a loss in structural stability, and repeated cycles of muscle fiber degeneration and regeneration. The satellite cells are the key stem cells responsible for muscle repair and reside between the basal lamina and sarcolemma. In this study, we aimed to determine whether pathological changes associated with the dystroglycanopathies affect satellite cell function. In the Large(myd) mouse dystroglycanopathy model, satellite cells are present in significantly greater numbers but display reduced proliferation on their native muscle fibers in vitro, compared with wild type. However, when removed from their fiber, proliferation in culture is restored to that of wild type. Immunohistochemical analysis of Large(myd) muscle reveals alterations to the basal lamina and interstitium, including marked disorganization of laminin, upregulation of fibronectin and collagens. Proliferation and differentiation of wild-type satellite cells is impaired when cultured on substrates such as collagen and fibronectin, compared with laminins. When engrafted into irradiated tibialis anterior muscles of mdx-nude mice, wild-type satellite cells expanded on laminin contribute significantly more to muscle regeneration than those expanded on fibronectin. These results suggest that defects in α-dystroglycan glycosylation are associated with an alteration in the satellite cell niche, and that regenerative potential in the dystroglycanopathies may be perturbed.

The dystrophin-glycoprotein complex in brain development and disease.

In addition to muscle disease, defects in processing and assembly of the dystrophin-glycoprotein complex (DGC) are associated with a spectrum of brain abnormalities ranging from mild cognitive impairment (MCI) to neuronal migration disorders. In brain, the DGC is involved in the organisation of GABA(A) receptors (GABA(A)Rs) and aquaporin-4 (AQP4)-containing protein complexes in neurons and glia, respectively. During development, defects in the glycosylation of α-dystroglycan that impair its ability to interact with the extracellular matrix (ECM) are frequently associated with cobblestone lissencephaly and mental retardation. Furthermore, mutations in the gene encoding ɛ-sarcoglycan (SGCE) cause the neurogenic movement disorder myoclonus dystonia syndrome. In this review, we describe recent progress in defining distinct roles for the DGC in neurons and glia.

Fukutin-related protein alters the deposition of laminin in the eye and brain.

Mutations in fukutin-related protein (FKRP) are responsible for a common group of muscular dystrophies ranging from adult onset limb girdle muscular dystrophies to severe congenital forms with associated structural brain involvement. The defining feature of this group of disorders is the hypoglycosylation of α-dystroglycan and its inability to effectively bind extracellular matrix ligands such as laminin α2. However, α-dystroglycan has the potential to interact with a number of laminin isoforms many of which are basement membrane/tissue specific and developmentally regulated. To further investigate this we evaluated laminin α-chain expression in the cerebral cortex and eye of our FKRP knock-down mouse (FKRP(KD)). These mice showed a marked disturbance in the deposition of laminin α-chains including α1, α2, α4, and α5, although only laminin α1- and γ1-chain mRNA expression was significantly upregulated relative to controls. Moreover, there was a diffuse pattern of laminin deposition below the pial surface which correlated with an abrupt termination of many of the radial glial cells. This along with the pial basement membrane defects, contributed to the abnormal positioning of both early- and late-born neurons. Defects in the inner limiting membrane of the eye were associated with a reduction of laminin α1 demonstrating the involvement of the α-dystroglycan:laminin α1 axis in the disease process. These observations demonstrate for the first time that a reduction in Fkrp influences the ability of tissue-specific forms of α-dystroglycan to direct the deposition of several laminin isoforms in the formation of different basement membranes.

Muscular dystrophies due to glycosylation defects: diagnosis and therapeutic strategies.

PURPOSE OF REVIEW: Dystroglycanopathies are a common group of diseases characterized by a reduction in α-dystroglycan glycosylation. This review discusses the recent novel discovery of additional dystroglycanopathy variants and progress in dystroglycanopathy animal models. RECENT FINDINGS: Several novel glycosyltransferase genes have been found to be responsible for a dystroglycanopathy phenotype, and in addition recessive mutations in DAG1 have been identified for the first time in a primary dystroglycanopathy. Studies in dystroglycanopathy mouse models have clarified some aspects of the structural defects observed in the central nervous system and in the eye, whereas a study in zebrafish implicates unfolded protein response in the pathogenesis of two of the secondary dystroglycanopathies. SUMMARY: Improved understanding of the molecular bases of dystroglycanopathies will lead to more precise diagnosis and genetic counseling; therapeutic strategies are being developed and tested in the preclinical models and it is hoped that these observations will pave the way to therapeutic interventions in humans.

Second international workshop for glycosylation defects in muscular dystrophies, 11-12 November, 2010, Charlotte, USA.

The second International Workshop for Glycosylation Defects in Muscular Dystrophies took place on November 11 and 12, 2010 in Charlotte, North Carolina, USA. The meeting was hosted by the Carolinas Medical Center with financial support from the Carolinas Muscular Dystrophy Research Endowment at the Carolinas HealthCare Foundation, the Muscular Dystrophy Association and funds raised by the "Jeans, Genes & Geniuses" event organized by Jane and Luther Lockwood. Since conducting the first workshop in May 2008, significant progress has been made in a subset of muscular dystrophies associated with defects in alpha-dystroglycan (α-DG) glycosylation. New findings on α-DG glycosylation and creation of novel animal models have expanded our understanding of the disease mechanism. The 2010 workshop focused on the following topics; (i) functional glycosylation of α-DG; (ii) animal models; and (iii) novel experimental therapies. The workshop brought together a total of 22 internationally renowned scientists and clinicians from US, UK, Denmark and Japan with active research and expertise in these areas. Overall, the workshop provided a unique opportunity to discuss the significance of recent progress, facilitate international collaboration, and identify new approaches to treat the disease.

Transgenic overexpression of LARGE induces α-dystroglycan hyperglycosylation in skeletal and cardiac muscle.

BACKGROUND: LARGE is one of seven putative or demonstrated glycosyltransferase enzymes defective in a common group of muscular dystrophies with reduced glycosylation of α-dystroglycan. Overexpression of LARGE induces hyperglycosylation of α-dystroglycan in both wild type and in cells from dystroglycanopathy patients, irrespective of their primary gene defect, restoring functional glycosylation. Viral delivery of LARGE to skeletal muscle in animal models of dystroglycanopathy has identical effects in vivo, suggesting that the restoration of functional glycosylation could have therapeutic applications in these disorders. Pharmacological strategies to upregulate Large expression are also being explored. METHODOLOGY/PRINCIPAL FINDINGS: In order to asses the safety and efficacy of long term LARGE over-expression in vivo, we have generated four mouse lines expressing a human LARGE transgene. On observation, LARGE transgenic mice were indistinguishable from the wild type littermates. Tissue analysis from young mice of all four lines showed a variable pattern of transgene expression: highest in skeletal and cardiac muscles, and lower in brain, kidney and liver. Transgene expression in striated muscles correlated with α-dystroglycan hyperglycosylation, as determined by immunoreactivity to antibody IIH6 and increased laminin binding on an overlay assay. Other components of the dystroglycan complex and extracellular matrix ligands were normally expressed, and general muscle histology was indistinguishable from wild type controls. Further detailed muscle physiological analysis demonstrated a loss of force in response to eccentric exercise in the older, but not in the younger mice, suggesting this deficit developed over time. However this remained a subclinical feature as no pathology was observed in older mice in any muscles including the diaphragm, which is sensitive to mechanical load-induced damage. CONCLUSIONS/SIGNIFICANCE: This work shows that potential therapies in the dystroglycanopathies based on LARGE upregulation and α-dystroglycan hyperglycosylation in muscle should be safe.

Altered primary and secondary myogenesis in the myostatin-null mouse.

Skeletal muscle fiber generation occurs principally in two myogenic phases: (1) Primary (embryonic) myogenesis when myoblasts proliferate and fuse to form primary myotubes and (2) secondary (fetal) myogenesis when successive waves of myoblasts fuse along the surface of the primary myotubes, giving rise to a population of smaller and more numerous secondary myotubes. This sequence of events determines fiber number and is completed at or soon after birth in most muscles of the mouse. The adult myostatin null mouse (MSTN(-/-)) displays both an increase in fiber number and size relative to wild type (MSTN(+/+)), suggesting a developmental origin for the hypermuscular phenotype. The focus of the present study was to determine at which point during myogenesis do MSTN(-/-) animals diverge from MSTN(+/+). To achieve this, we focused on the extensor digitorum longus (EDL) muscle and evaluated primary myotube number at embryonic day (E) 13.0 and E14.5 and secondary to primary myotube ratios at E18.5. We show that primary myotube number and size were significantly increased in the MSTN(-/-) mice by E14.5 and the secondary to primary myotube ratio increased at E18.5. This increase in the rate of fiber formation resulted in MSTN(-/-) mice harboring 87% of their final adult fiber number at E18.5, compared to only 73% in MSTN(+/+). An accelerated myogenic program in the MSTN(-/-) mice was further confirmed by our finding of an initial expansion in the myogenic stem cell (identified through Pax7 expression) and myoblast (identified through myogenin expression) cell pools at E14.5 in the EDL muscle of these animals that was, however, followed by a reduction of both populations of cells at E18.5 relative to MSTN(+/+). Overall these data suggest that the genetic loss of myostatin accelerates the developmental myogenic program of primary and secondary skeletal myogenesis.

A comparative study of alpha-dystroglycan glycosylation in dystroglycanopathies suggests that the hypoglycosylation of alpha-dystroglycan does not consistently correlate with clinical severity.

Hypoglycosylation of alpha-dystroglycan underpins a subgroup of muscular dystrophies ranging from congenital onset of weakness, severe brain malformations and death in the perinatal period to mild weakness in adulthood without brain involvement. Mutations in six genes have been identified in a proportion of patients. POMT1, POMT2 and POMGnT1 encode for glycosyltransferases involved in the mannosylation of alpha-dystroglycan but the function of fukutin, FKRP and LARGE is less clear. The pathological hallmark is reduced immunolabeling of skeletal muscle with antibodies recognizing glycosylated epitopes on alpha-dystroglycan. If the common pathway of these conditions is the hypoglycosyation of alpha-dystroglycan, one would expect a correlation between clinical severity and the extent of hypoglycosylation. By studying 24 patients with mutations in these genes, we found a good correlation between reduced alpha-dystroglycan staining and clinical course in patients with mutations in POMT1, POMT2 and POMGnT1. However, this was not always the case in patients with defects in fukutin and FKRP, as we identified patients with mild limb-girdle phenotypes without brain involvement with profound depletion of alpha-dystroglycan. These data indicate that it is not always possible to correlate clinical course and alpha-dystroglycan labeling and suggest that there might be differences in alpha-dystroglycan processing in these disorders.

Assessment of the transformation of equine skin-derived fibroblasts to multinucleated skeletal myotubes following lentiviral-induced expression of equine myogenic differentiation 1.

OBJECTIVE: To develop a reliable method for converting cultured equine skin-derived fibroblasts into muscle cells. SAMPLE POPULATION: Equine skin-derived fibroblasts. PROCEDURES: The equine myogenic differentiation 1 (eqMyoD) genomic sequence was obtained by use of equine bacterial artificial chromosome screening and PCR sequencing. Total mRNA was extracted from foal skeletal muscle, and eqMyoD cDNA was cloned into a plasmid vector with an internal ribosomal entry site to express bicistronic eqMyoD or enhanced green fluorescent protein (EGFP). Transient expression was confirmed by immunocytochemical analysis and western immunoblots in equine fibroblasts and fibroblasts from National Institutes of Health Swiss mouse embryos, prior to generation of a lentiviral vector containing the same coding sequences. Transformation of equine skin-derived cells into skeletal myotubes was examined by use of immunohistochemical analysis, western immunoblotting, and periodic acid-Schiff staining. RESULTS: eqMyoD mRNA consists of 960 bp and shares high homology with myogenic differentiation 1 from other mammals. Transfection confirmed the expression of a 53-kd protein with mainly nuclear localization. Lentiviral transduction was efficient, with approximately 80% of EGFP-positive cells transformed into multinucleated myotubes during 15 days, as determined by expression of the muscle-specific proteins desmin, troponin-T, and sarcomeric myosin and by cytoplasmic storage of glycogen. CONCLUSIONS AND CLINICAL RELEVANCE: Equine primary fibroblasts were transformed by lentiviral transduction of eqMyoD into fusion-competent myoblasts. This may offer a preferable alternative to primary myoblast cultures for the investigation of cellular defects associated with muscle diseases of horses, such as recurrent exertional rhabdomyolysis and polysaccharide storage myopathy.

Investigating the pathology of Emery-Dreifuss muscular dystrophy.

EDMD (Emery-Dreifuss muscular dystrophy) is caused by mutations in either the gene encoding for lamin A/C (LMNA) located at 1q21.2-q21.3 or emerin (EMD) located at Xq28. Autosomal dominant EDMD caused by LMNA mutations is more common than the X-linked form and often more severe, with an earlier onset. At the histological and histochemical levels, both X-linked and autosomal dominant EDMD appear similar. However, individuals with the same genetic disorder often show remarkable differences in clinical severity, a finding generally attributed to the genetic background. The clinical and pathological findings in EDMD patients found to have mutations in more than one gene are also discussed. There is now much interest in the phenotype of several animal models for EDMD which should lead to an increased insight into the pathogenesis of this disorder, particularly that relating to the heart phenotype.

Mild POMGnT1 mutations underlie a novel limb-girdle muscular dystrophy variant.

BACKGROUND: Mutations in protein-O-mannose-beta1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) have been found in muscle-eye-brain disease, a congenital muscular dystrophy with structural eye and brain defects and severe mental retardation. OBJECTIVE: To investigate whether mutations in POMGnT1 could be responsible for milder allelic variants of muscular dystrophy. DESIGN: Screening for mutations in POMGnT1. SETTING: Tertiary neuromuscular unit. PATIENT: A patient with limb-girdle muscular dystrophy phenotype, with onset at 12 years of age, severe myopia, normal intellect, and decreased alpha-dystroglycan immunolabeling in skeletal muscle. RESULTS: A homozygous POMGnT1 missense mutation (c.1666G>A, p.Asp556Asn) was identified. Enzyme studies of the patient's fibroblasts showed an altered kinetic profile, less marked than in patients with muscle-eye-brain disease and in keeping with the relatively mild phenotype in our patient. CONCLUSIONS: Our findings widen the spectrum of disorders known to result from mutations in POMGnT1 to include limb-girdle muscular dystrophy with no mental retardation. We propose that this condition be known as LGMD2M. The enzyme assay used to diagnose muscle-eye-brain disease may not detect subtle abnormalities of POMGnT1 function, and additional kinetic studies must be carried out in such cases.