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.

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.

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.

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.

Desmin immunolocalisation in autosomal dominant Emery-Dreifuss muscular dystrophy.

Autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD) is one of a number of allelic disorders caused by mutations in the nuclear lamina proteins, lamins A and C. The disorder is characterised by the early onset of skeletal muscle weakness and joint contractures and later, by dilated cardiomyopathy and cardiac arrythmias. Although the pathophysiology is not understood, one theory suggests that disordered structural organisation at weakened nuclei in contractile cells may underlie the disease. Previous work shows that mice deficient in lamin A/C develop similar skeletal and cardiac muscle signs to patients with AD-EDMD and ultrastructural examination of muscle from these mice shows abnormal localisation of desmin. We hypothesised therefore that desmin localisation may be abnormal in muscle or cells from patients with AD-EDMD and/or in cells expressing mutant lamins. In order to evaluate this, desmin immunolocalisation was determined in skeletal muscle biopsy sections from patients with AD-EDMD and cell lines including MyoD-transfected fibroblast-derived myotubes from AD-EDMD patients and murine embryonic stem cell-derived cardiomyocytes stably transfected with mutant human lamin A. Ultrastructural examination of patient muscle was also performed. Desmin was expressed and localised normally in patient muscle and cell lines and ultrastructural examination was similar to controls. These results fail to provide any evidence that dominant mutations in lamin A/C lead to a disorganisation of the desmin associated cytoskeleton.

Sub-cellular localisation of fukutin related protein in different cell lines and in the muscle of patients with MDC1C and LGMD2I.

MDC1C and LGMD2I are two allelic forms of muscular dystrophies caused by mutations in the gene encoding for fukutin related protein (FKRP). FKRP encodes for a putative glycosyltransferase, the precise function of which is unknown. However, the marked reduction of alpha-dystroglycan glycosylation in the muscle of MDC1C and LGMD2I patients suggests a role for FKRP in dystroglycan processing. Using a polyclonal antibody raised against FKRP we now show that endogenous FKRP locates to the Golgi apparatus of neuronal, oligodendroglial, and the cardiac muscle cell line H9c2. In differentiated C2C12 myotubes and in transverse sections of normal skeletal and cardiac muscle, endogenous FKRP surrounded the myonuclei. This localisation was unaffected in the skeletal muscle of patients with MDC1C and LGMD2I carrying various FKRP mutations. These observations imply a specific role for FKRP during striated muscle, neuronal and glial development and suggest that protein mis-localisation is not a common mechanism of disease in FKRP-related dystrophies.

Syncoilin upregulation in muscle of patients with neuromuscular disease.

Syncoilin may have a role in linking the desmin-associated intermediate filament network of the muscle fiber with the dystrophin-associated protein complex (DAPC). We have evaluated syncoilin in a range of neuromuscular disorders including Duchenne and Becker muscular dystrophy, central core disease, congenital muscular dystrophies, and neurogenic disorders. Our results show that syncoilin immunolabeling is not only altered in muscle fibers with alterations in the DAPC but also in response to a variety of genetic defects, including those associated with proteins of the extracellular matrix and the intracellular Ca2+-release channel (ryanodine receptor). The pattern of syncoilin immunolabeling in these diseases appeared to reflect a rearrangement of the intermediate filament-associated cytoskeleton that characterizes both muscle fiber development and conditions in which the cytoskeletal organization of the muscle fiber is significantly affected. These observations raise the possibility that mutations in the gene encoding for syncoilin may underlie some forms of muscle disease.

Ullrich congenital muscular dystrophy: connective tissue abnormalities in the skin support overlap with Ehlers-Danlos syndromes.

Ullrich congenital muscular dystrophy (UCMD) is caused by mutations in the three genes coding for the alpha chains of collagen VI and characterized by generalized muscle weakness, striking hypermobility of distal joints in conjunction with variable contractures of more proximal joints, and normal intellectual development. The diagnosis is supported by abnormal immunoreactivity for collagen VI on muscle biopsies. As patients with UCMD show clinical characteristics typical of classical disorders of connective tissue such as Ehlers-Danlos syndromes (EDS), we investigated the ultrastructure of skin biopsy samples from patients with UCMD (n=5). Electron microscopy of skin biopsies revealed ultrastructural abnormalities in all cases, including alterations of collagen fibril morphology (variation in size and composite fibers) and increase in ground substance, which resemble those seen in patients with EDS. Our findings suggest that there is a true connective tissue component as part of the phenotypic spectrum of UCMD and that there is considerable clinical as well as morphological overlap between UCMD and classic connective tissue disorders.

Prenatal diagnosis of Ullrich congenital muscular dystrophy using haplotype analysis and collagen VI immunocytochemistry.

OBJECTIVES: Ullrich congenital muscular dystrophy (UCMD) is a recessively inherited condition characterised by proximal joint contractures, marked distal joint hyperextensibility, rigidity of the spine and early respiratory failure. Recently, mutations in the genes encoding the subunits of collagen VI have been identified in this disease. We undertook two prenatal diagnoses for UCMD in a consanguineous family where the disease was consistent with linkage to the COL6A3 locus and immunolabelling of collagen VI in the proband's skeletal muscle was severely reduced. METHODS: Both haplotype analysis and collagen VI immunolabelling were used to determine the status of the fetuses. RESULTS: Haplotype analysis of DNA extracted from chorionic villus samples (CVS) from the initial at-risk pregnancy with markers encompassing COL6A3 demonstrated that this fetus had inherited the same haplotypes as the affected child, and immunolabelling of the at-risk CVS demonstrated the virtual absence of collagen VI. A second latter fetus inherited neither of the at-risk haplotypes and collagen VI expression in the CVS was normal. During the second pregnancy, a homozygous G > A change in the last nucleotide of exon 27 of COL6A3 was identified in the proband, substantiating the results obtained from haplotype analysis and collagen VI immunolabelling. CONCLUSION: These findings demonstrate that haplotype analysis in combination with immunocytochemistry is a rapid and reliable method for prenatal diagnosis of UCMD, provided the family is genetically informative and reduced collagen VI expression in the proband has been demonstrated.

Enhanced expression of the P2X4 receptor in Duchenne muscular dystrophy correlates with macrophage invasion.

Following molecular and immunohistochemical analysis of the purinergic P2X4 receptor subunit in dystrophin-deficient muscle we have identified a distinct subpopulation of P2X4-positive cells infiltrating the dystrophic fibres. These cells were absent from normal muscle and rarely present in the dystrophic muscle taken before and after the onset of degeneration. We have identified these P2X4-positive cells as macrophages, demonstrating for the first time that human and mouse tissue macrophages express P2X4 in addition to P2X7 receptor subunits both in vitro and in situ. Moreover, we have demonstrated that the increase in the P2X4 expression is yet another feature of an inflammatory response identified in DNA arrays of dystrophic muscle. Immunohistochemical analysis failed to localise discernible expression of P2X4 protein in adult skeletal or cardiac muscle fibres, whilst myoblasts in culture expressed low levels of this subunit, as detected by RT-PCR and Western blotting. In light of the involvement of macrophages in the dystrophic process, the function of P2X receptors and their role in the Duchenne pathology as well as their potential role in therapeutic applications are discussed.

Mutations in the human LARGE gene cause MDC1D, a novel form of congenital muscular dystrophy with severe mental retardation and abnormal glycosylation of alpha-dystroglycan.

The congenital muscular dystrophies (CMD) are a heterogeneous group of autosomal recessive disorders. A new pathomechanism has recently been identified in a group of these disorders in which known or putative glycosyltransferases are defective. Common to all these conditions is the hypoglycosylation of alpha-dystroglycan. Fukuyama CMD, muscle-eye-brain disease and Walker-Warburg syndrome, each associated with eye abnormalities and neuronal migration defects, result from mutations in fukutin, POMGnT1 and POMT1, respectively, while mutations in the fukutin-related protein (FKRP) gene cause congenital muscular dystrophy 1C, typically lacking brain involvement. Another putative glycosyltransferase, Large, is mutated in the myodystrophy mouse. The human homologue of this gene is therefore a strong candidate for involvement in novel forms of muscular dystrophy. We studied 36 patients with muscular dystrophy and either mental retardation, structural brain changes or abnormal alpha-dystroglycan immunolabelling, unlinked to any reported CMD loci. Linkage analysis in seven informative families excluded involvement of LARGE but sequencing of this gene in the remaining 29 families identified one patient with a G1525A (Glu509Lys) missense mutation and a 1 bp insertion, 1999insT. This 17-year-old girl presented with congenital muscular dystrophy, profound mental retardation, white matter changes and subtle structural abnormalities on brain MRI. Her skeletal muscle biopsy showed reduced immunolabelling of alpha-dystroglycan. Immunoblotting with an antibody to a glycosylated epitope demonstrated a reduced molecular weight form of alpha-dystroglycan that retained some laminin binding activity. This is the first description of mutations in the human LARGE gene and we propose to name this new disorder MDC1D.

Phenotypic spectrum associated with mutations in the fukutin-related protein gene.

We describe 22 patients with mutations in the fukutin-related protein (FKPR) gene. Four patients had congenital muscular dystrophy (MDC1C), with presentation at birth, severe weakness and inability to stand unsupported. The other 18 had limb girdle muscular dystrophy (LGMD2I). Eleven showed a Duchenne-like course with loss of ambulation in the early teens while 7 had a milder phenotype. Muscle biopsy invariably showed abnormal expression of a-dystroglycan. MDC1C patients either carried 2 missense or 1 missense and 1 nonsense mutations. Patients with LGMD2I shared a common mutation (C826A,Leu276Ileu) and their phenotypic severity was correlated with the second allelic mutation.

Muscle MRI findings in a three-generation family affected by Bethlem myopathy.

We report clinical and muscle magnetic resonance imaging (MRI) findings in three individuals (aged 6, 26 and 73 years) from a three-generation family with Bethlem myopathy, confirmed by molecular genetic analysis which showed an exon skipping mutation in the COL6A1 gene. The clinical severity ranged from mild proximal weakness and distal laxity in the younger patients, to inability to stand or walk and severe contractures in the 76-year-old grandmother. The pattern of muscle involvement showed variable severity in parallel with the severity of motor function impairment. Although there was a marked variability in the severity of the MRI findings, it was possible to recognize a specific pattern of muscle involvement in all three patients. This consisted of involvement of the peripheral region of the vastus lateralis and hamstrings muscles with relative sparing of their central part. This was best appreciated in the third decade of life, but could also be identified both in the younger patient with minimal MRI changes and in the oldest patient, despite her more severe and diffuse muscle involvement. This report suggests that muscle MRI could be used as an additional tool to establish the pattern and the degree of muscle involvement in patients with Bethlem myopathy. Further studies in a larger cohort are needed to evaluate the specificity of these findings.