Changes In Dystrophic Muscles Research Articles

Magnetic-field-driven targeting of exosomes modulates immune and metabolic changes in dystrophic muscle

Exosomes are promising therapeutics for tissue repair and regeneration to induce and guide appropriate immune responses in dystrophic pathologies. However, manipulating exosomes to control their biodistribution and targeting them in vivo to achieve adequate therapeutic benefits still poses a major challenge. Here we overcome this limitation by developing an externally controlled delivery system for primed annexin A1 myo-exosomes (Exomyo). Effective nanocarriers are realized by immobilizing the Exomyo onto ferromagnetic nanotubes to achieve controlled delivery and localization of Exomyo to skeletal muscles by systemic injection using an external magnetic field. Quantitative muscle-level analyses revealed that macrophages dominate the uptake of Exomyo from these ferromagnetic nanotubes in vivo to synergistically promote beneficial muscle responses in a murine animal model of Duchenne muscular dystrophy. Our findings provide insights into the development of exosome-based therapies for muscle diseases and, in general, highlight the formulation of effective functional nanocarriers aimed at optimizing exosome biodistribution.

Collagen cross-links scale with passive stiffness in dystrophic mouse muscles, but are not altered with administration of a lysyl oxidase inhibitor.

In Duchenne muscular dystrophy (DMD), a lack of functional dystrophin leads to myofiber instability and progressive muscle damage that results in fibrosis. While fibrosis is primarily characterized by an accumulation of extracellular matrix (ECM) components, there are changes in ECM architecture during fibrosis that relate more closely to functional muscle stiffness. One of these architectural changes in dystrophic muscle is collagen cross-linking, which has been shown to increase the passive muscle stiffness in models of fibrosis including the mdx mouse, a model of DMD. We tested whether the intraperitoneal injections of beta-aminopropionitrile (BAPN), an inhibitor of the cross-linking enzyme lysyl oxidase, would reduce collagen cross-linking and passive stiffness in young and adult mdx mice compared to saline-injected controls. We found no significant differences between BAPN treated and saline treated mice in collagen cross-linking and stiffness parameters. However, we observed that while collagen cross-linking and passive stiffness scaled positively in dystrophic muscles, collagen fiber alignment scaled with passive stiffness distinctly between muscles. We also observed that the dystrophic diaphragm showed the most dramatic fibrosis in terms of collagen content, cross-linking, and stiffness. Overall, we show that while BAPN was not effective at reducing collagen cross-linking, the positive association between collagen cross-linking and stiffness in dystrophic muscles still show cross-linking as a viable target for reducing passive muscle stiffness in DMD or other fibrotic muscle conditions.

Peripheral blood transcriptome profiling enables monitoring disease progression in dystrophic mice and patients

DMD is a rare disorder characterized by progressive muscle degeneration and premature death. Therapy development is delayed by difficulties to monitor efficacy non‐invasively in clinical trials. In this study, we used RNA‐sequencing to describe the pathophysiological changes in skeletal muscle of 3 dystrophic mouse models. We show how dystrophic changes in muscle are reflected in blood by analyzing paired muscle and blood samples. Analysis of repeated blood measurements followed the dystrophic signature at five equally spaced time points over a period of seven months. Treatment with two antisense drugs harboring different levels of dystrophin recovery identified genes associated with safety and efficacy. Evaluation of the blood gene expression in a cohort of DMD patients enabled the comparison between preclinical models and patients, and the identification of genes associated with physical performance, treatment with corticosteroids and body measures. The presented results provide evidence that blood RNA‐sequencing can serve as a tool to evaluate disease progression in dystrophic mice and patients, as well as to monitor response to (dystrophin‐restoring) therapies in preclinical drug development and in clinical trials.

Multiomics analysis of the mdx/mTR mouse model of Duchenne muscular dystrophy

ABSTRACT Purpose/Aim Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disease characterized by extensive muscle weakness. Patients with DMD lack a functional dystrophin protein, which transmits force and organizes the cytoskeleton of skeletal muscle. Multiomic studies have been proposed as a way to obtain novel insight about disease processes from preclinical models, and we used this approach to study pathological changes in dystrophic muscles. Materials and Methods We evaluated hindlimb muscles of male mdx/mTR mice, which lack a functional dystrophin protein and have deficits in satellite cell abundance and proliferative capacity. Wild type (WT) C57BL/6 J mice served as controls. Muscle fiber contractility was measured, along with changes in the transcriptome using RNA sequencing, and in the proteome, metabolome, and lipidome using mass spectrometry. Results While mdx/mTR mice displayed gross pathological changes and continued cycles of degeneration and regeneration, we found no differences in permeabilized fiber contractility between strains. However, there were numerous changes in the transcriptome and proteome related to protein balance, contractile elements, extracellular matrix, and metabolism. There was only a 53% agreement in fold-change data between the proteome and transcriptome. Numerous changes in markers of skeletal muscle metabolism were observed, with dystrophic muscles exhibiting elevated glycolytic metabolites such as 6-phosphoglycerate, fructose-6-phosphate and glucose-6-phosphate, fructose bisphosphate, phosphorylated hexoses, and phosphoenolpyruvate. Conclusions These findings highlight the utility of multiomics in studying muscle disease, and provide additional insight into the pathological changes in dystrophic muscles that might help to indirectly guide evidence-based nutritional or exercise prescription in DMD patients.

Lectin-binding characterizes the healthy human skeletal muscle glycophenotype and identifies disease-specific changes in dystrophic muscle.

Our understanding of muscle glycosylation to date has derived from studies in mouse models and a limited number of human lectin histochemistry studies. As various therapeutic approaches aimed at treating patients with muscular dystrophies are being translated from rodent models to human, it is critical to better understand human muscle glycosylation and relevant disease-specific differences between healthy and dystrophic muscle. Here, we report the first quantitative characterization of human muscle glycosylation, and identify differentiation- and disease-specific differences in human muscle glycosylation. Utilizing a panel of 13 lectins with varying glycan specificities, we surveyed lectin binding to primary and immortalized myoblasts and myotubes from healthy and dystrophic sources. Following differentiation of primary and immortalized healthy human muscle cells, we observed increased binding of Narcissus pseudonarcissus agglutinin (NPA), PNA, MAA-II and WFA to myotubes compared to myoblasts. Following differentiation of immortalized healthy and dystrophic human muscle cells, we observed disease-specific differences in binding of NPA, Jac and Tricosanthes japonica agglutinin-I (TJA-I) to differentiated myotubes. We also observed differentiation- and disease-specific differences in binding of NPA, Jac, PNA, TJA-I and WFA to glycoprotein receptors in muscle cells. Additionally, Jac, PNA and WFA precipitated functionally glycosylated α-DG, that bound laminin, while NPA and TJA-I did not. Lectin histochemistry of healthy and dystrophic human muscle sections identified disease-specific differences in binding of O-glycan and sialic acid-specific lectins between healthy and dystrophic muscle. These results indicate that specific and discrete changes in glycosylation occur following differentiation, and identify specific lectins as potential biomarkers sensitive to changes in healthy human muscle glycosylation.

Comparative Label-Free Mass Spectrometric Analysis of Mildly versus Severely Affected mdx Mouse Skeletal Muscles Identifies Annexin, Lamin, and Vimentin as Universal Dystrophic Markers.

The primary deficiency in the membrane cytoskeletal protein dystrophin results in complex changes in dystrophic muscles. In order to compare the degree of secondary alterations in differently affected subtypes of skeletal muscles, we have conducted a global analysis of proteome-wide changes in various dystrophin-deficient muscles. In contrast to the highly degenerative mdx diaphragm muscle, which showed considerable alterations in 35 distinct proteins, the spectrum of mildly to moderately dystrophic skeletal muscles, including interosseus, flexor digitorum brevis, soleus, and extensor digitorum longus muscle, exhibited a smaller number of changed proteins. Compensatory mechanisms and/or cellular variances may be responsible for differing secondary changes in individual mdx muscles. Label-free mass spectrometry established altered expression levels for diaphragm proteins associated with contraction, energy metabolism, the cytoskeleton, the extracellular matrix and the cellular stress response. Comparative immunoblotting verified the differences in the degree of secondary changes in dystrophin-deficient muscles and showed that the up-regulation of molecular chaperones, the compensatory increase in proteins of the intermediate filaments, the fibrosis-related increase in collagen levels and the pathophysiological decrease in calcium binding proteins is more pronounced in mdx diaphragm as compared to the less severely affected mdx leg muscles. Annexin, lamin, and vimentin were identified as universal dystrophic markers.

Effect of Nuclear Factor κB Inhibition on Serotype 9 Adeno-Associated Viral (AAV9) Minidystrophin Gene Transfer to the mdx Mouse

Gene therapy studies for Duchenne muscular dystrophy (DMD) have focused on viral vector-mediated gene transfer to provide therapeutic protein expression or treatment with drugs to limit dystrophic changes in muscle. The pathological activation of the nuclear factor (NF)-κB signaling pathway has emerged as an important cause of dystrophic muscle changes in muscular dystrophy. Furthermore, activation of NF-κB may inhibit gene transfer by promoting inflammation in response to the transgene or vector. Therefore, we hypothesized that inhibition of pathological NF-κB activation in muscle would complement the therapeutic benefits of dystrophin gene transfer in the mdx mouse model of DMD. Systemic gene transfer using serotype 9 adeno-associated viral (AAV9) vectors is promising for treatment of preclinical models of DMD because of vector tropism to cardiac and skeletal muscle. In quadriceps of C57BL/10ScSn-Dmd(mdx)/J (mdx) mice, the addition of octalysine (8K)-NF-κB essential modulator (NEMO)-binding domain (8K-NBD) peptide treatment to AAV9 minidystrophin gene delivery resulted in increased levels of recombinant dystrophin expression suggesting that 8K-NBD treatment promoted an environment in muscle tissue conducive to higher levels of expression. Indices of necrosis and regeneration were diminished with AAV9 gene delivery alone and to a greater degree with the addition of 8K-NBD treatment. In diaphragm muscle, high-level transgene expression was achieved with AAV9 minidystoophin gene delivery alone; therefore, improvements in histological and physiological indices were comparable in the two treatment groups. The data support benefit from 8K-NBD treatment to complement gene transfer therapy for DMD in muscle tissue that receives incomplete levels of transduction by gene transfer, which may be highly significant for clinical applications of muscle gene delivery.

Quantitative muscle ultrasound is a promising longitudinal follow-up tool in Duchenne muscular dystrophy

Responsive outcome measures are needed to follow the disease status of Duchenne muscular dystrophy (DMD) patients, as new therapeutic approaches become available for affected boys. Quantitative muscle ultrasound (QMUS) is potentially an attractive follow up tool for DMD because it reflects the severity of the dystrophic process without the need for invasive procedures, by quantifying echo intensity (i.e. mean grey level of muscle images) and muscle thickness. We performed a longitudinal follow-up of lower and upper extremity QMUS in 18 DMD patients and compared this with physical functioning in 11 of these patients. QMUS could be performed in every patient, and no patient was subjected to more than a total of 20min of ultrasound scanning time for this study. As expected we found a significant increase of echo intensity with age, reflecting increasing dystrophic muscle changes. This increase was related to ambulatory status, functional grading, muscle strength and motor ability. Our study establishes QMUS as a practical and child-friendly tool for the longitudinal follow up of DMD patients.

AAV6-mediated Systemic shRNA Delivery Reverses Disease in a Mouse Model of Facioscapulohumeral Muscular Dystrophy

Treatment of dominantly inherited muscle disorders remains a difficult task considering the need to eliminate the pathogenic gene product in a body-wide fashion. We show here that it is possible to reverse dominant muscle disease in a mouse model of facioscapulohumeral muscular dystrophy (FSHD). FSHD is a common form of muscular dystrophy associated with a complex cascade of epigenetic events following reduction in copy number of D4Z4 macrosatellite repeats located on chromosome 4q35. Several 4q35 genes have been examined for their role in disease, including FRG1. Overexpression of FRG1 causes features related to FSHD in transgenic mice and the FRG1 mouse is currently the only available mouse model of FSHD. Here we show that systemic delivery of RNA interference expression cassettes in the FRG1 mouse, after the onset of disease, led to a dose-dependent long-term FRG1 knockdown without signs of toxicity. Histological features including centrally nucleated fibers, fiber size reduction, fibrosis, adipocyte accumulation, and inflammation were all significantly improved. FRG1 mRNA knockdown resulted in a dramatic restoration of muscle function. Through RNA interference (RNAi) expression cassette redesign, our method is amenable to targeting any pathogenic gene offering a viable option for long-term, body-wide treatment of dominant muscle disease in humans.

A single 30 min treadmill exercise session is suitable for ‘proof-of concept studies’ in adult mdx mice: A comparison of the early consequences of two different treadmill protocols

The extent of muscle pathology in sedentary adult mdx mice is very low and treadmill exercise is often used to increase myofibre necrosis; however, the early events in dystrophic muscle and blood in response to treadmill exercise (leading to myofibre necrosis) are unknown. This study describes in detail two standardised protocols for the treadmill exercise of mdx mice and profiles changes in molecular and cellular events after a single 30min treadmill session (Protocol A) or after 4weeks of (twice weekly) treadmill exercise (Protocol B). Both treadmill protocols increased multiple markers of muscle damage. We conclude that a single 30min treadmill exercise session is a sufficient and conveniently fast screening test and could be used in ‘proof-of-concept’ studies to evaluate the benefits of pre-clinical drugs in vivo. Myofibre necrosis, blood serum CK and oxidative stress (specifically the ratio of oxidised to reduced protein thiols) are reliable markers of muscle damage after exercise; many parameters demonstrated high biological variation including changes in mRNA levels for key inflammatory cytokines in muscle. The sampling (sacrifice and tissue collection) time after exercise for these parameters is critical. A more precise understanding of the changes in dystrophic muscle after exercise aims to identify biomarkers and new potential therapeutic drug targets for Duchenne Muscular Dystrophy.

Mutation in Exon 1f of PLEC, Leading to Disruption of Plectin Isoform 1f, Causes Autosomal-Recessive Limb-Girdle Muscular Dystrophy

Limb-girdle muscular dystrophy (LGMD) is a genetically heterogeneous group of inherited muscular disorders manifesting symmetric, proximal, and slowly progressive muscle weakness. Using Affymetrix 250K SNP Array genotyping and homozygosity mapping, we mapped an autosomal-recessive LGMD phenotype to the telomeric portion of chromosome 8q in a consanguineous Turkish family with three affected individuals. DNA sequence analysis of PLEC identified a homozygous c.1_9del mutation containing an initiation codon in exon 1f, which is an isoform-specific sequence of plectin isoform 1f. The same homozygous mutation was also detected in two additional families during the analysis of 72 independent LGMD2-affected families. Moreover, we showed that the expression of PLEC was reduced in the patient's muscle and that there was almost no expression for plectin 1f mRNA as a result of the mutation. In addition to dystrophic changes in muscle, ultrastructural alterations, such as membrane duplications, an enlarged space between the membrane and sarcomere, and misalignment of Z-disks, were observed by transmission electron microscopy. Unlike the control skeletal muscle, no sarcolemmal staining of plectin was detected in the patient's muscle. We conclude that as a result of plectin 1f deficiency, the linkage between the sarcolemma and sarcomere is broken, which could affect the structural organization of the myofiber. Our data show that one of the isoforms of plectin plays a key role in skeletal muscle function and that disruption of the plectin 1f can cause the LGMD2 phenotype without any dermatologic component as was previously reported with mutations in constant exons of PLEC.

Detecting Degenerative Changes in Myotonic Murine Models of Duchenne Muscular Dystrophy Using High-Frequency Ultrasound

Ultrasound imaging is an economical and noninvasive technique for studying musculoskeletal diseases such as Duchenne muscular dystrophy (DMD). Duchenne muscular dystrophy results from the loss of the cytoskeletal protein dystrophin. This in turn increases muscle susceptibility to injury, resulting in myofiber membrane leakage, inflammation, and degeneration. The purpose of this study was to detect dystrophic changes in muscle noninvasively. High-frequency ultrasound (HFU; 40 MHz) was used to obtain a resolution of 80 microm, which is not achievable with lower-frequency clinical scanners. Using HFU, we were able to visualize musculoskeletal abnormalities as hyperechoic lesions within the dystrophic muscle. To validate the imaging findings, fiducial markers were placed in close proximity to lesions under HFU guidance. The nature of the lesion was then investigated histologically. This was repeated in the lower limbs of 10 mdx (mutated dystrophin gene) mice, a transgenic murine model of DMD. The abnormalities in the dystrophic muscle consisted of large influxes of leukocytic infiltrates, fibrotic scars, and calcified lesions. Although macrophages and fibrosis are commonly noted in DMD, to our knowledge, the presence of intramuscular calcific necrosis in dystrophic muscle has not been reported. This novel dystrophic feature of muscle degeneration may be useful in longitudinal studies of murine DMD and regenerative studies.

Abnormal expression of proteoglycans in Ullrich's disease with collagen VI deficiency

Patients with Ullrich's disease have generalized muscle weakness, multiple contractures of the proximal joints, and hyperextensibility of the distal joints. Recently we found a marked reduction of fibronectin receptors in the skin and cultured fibroblasts of two patients with Ullrich's disease with collagen VI deficiency, and speculated that an abnormality of cell adhesion may be involved in the pathogenesis of the disease. In this study, we investigated the expression of proteoglycans and adhesion molecules in Ullrich's disease and other muscle diseases. We found a reduction of NG2 proteoglycan in the membrane of skeletal muscle but not in the skin in Ullrich's disease. By contrast, we found the upregulation of tenascin C in the extracellular matrix of skeletal muscle in Ullrich's disease. Our findings suggest that abnormal expression of proteoglycans and adhesion molecules may be involved in the pathogenesis of the dystrophic muscle changes in Ullrich's disease.

241. Lentivirus Mediated Dystrophin Expression in mdx Muscles

Top of pageAbstract To study candidate treatments for Duchenne muscular dystrophy, we have generated a series of lentiviral vectors that express various reporter genes and mini-dystrophin cassettes and tested their ability to transduce a variety of cell types in vitro and in vivo. Direct injection of lentiviral vectors into adult skeletal muscle resulted in significantly lower levels of gene expression than were obtained using either AAV or adenoviral vectors. Since an advantage of lentiviral vectors is stable expression of a transgene that has integrated into host genomic DNA, stem cells are considered a good target of lentiviral vectors. During myogenesis, activated-satellite cells or muscle progenitor cells proliferate in the muscle microenvironment. Most of these cells become differentiated and form myofibers, while small numbers of them are stored adjacent to myofibers as mitotically quiescent satellite cells for future muscle regeneration. Skeletal muscle of newborn mice may be a good target for lentiviral vectors, because of relatively high numbers of activated muscle progenitor cells contributing to muscle growth and satellite cell pools. Therefore, we tried targeting satellite cells or muscle progenitor cells using lentivirual vectors expressing dystrophin mini-gene. We demonstrated that relatively higher levels of transuction were obtained with intra-muscular injection into neonatal muscles, and that both muscle fibers and primary cultured satellite cells stably expressed a GFP marker gene. We also demonstrated that lentiviral mediated truncated mini-dystrophin expression in mdx muscles may be useful for correction of pathological changes in dystrophic muscle. These data suggest that myogenic stem cells can be stably transduced with lentiviral vectors and may contribute to stable muscle regeneration in dystrophic muscle by enabling continuous expression of mini-dystrophin, which may have implications for gene therapy of Duchenne muscular dystrophy.

Pathological characteristics of skeletal muscle in Ullrich's disease with collagen VI deficiency

Patients with Ullrich's disease have generalized muscle weakness, multiple contractures of the proximal joints and hyperextensibility of the distal joints. Recently, we found a deficiency of collagen VI protein in skeletal muscle from two patients with Ullrich's disease. In this study, we investigated immunohistochemically the expression of extracellular matrix proteins and various proteins, which are markers for regenerating muscle fibers. Although we have detected the reduction of collagen VI in Ullrich's disease with the two kinds of monoclonal antibodies for the different domains of collagen VI, the remaining immunoreactive material was different between them. This might suggest the presence of incomplete collagen VI protein in the muscle fibers. Furthermore, we found that very small muscle fibers in the patients with Ullrich's disease showed marked expression of desmin, neural cell adhesion molecule and neonatal myosin heavy chain, which is a characteristic finding of regenerating fibers, however, they showed poor expression of developmental myosin heavy chain and thrombomodulin. The present findings suggest that abnormal regeneration or maturation processes are involved in the pathogenesis of dystrophic muscle changes at least in the advanced stage of Ullrich's disease.

Viral vectors for gene transfer of micro-, mini-, or full-length dystrophin

Gene therapy for Duchenne muscular dystrophy will require methods to deliver gene constructs encoding functional versions of dystrophin to the vast majority of a patient's musculature. Obstacles to achieving these goals include identifying which forms of dystrophin would be effective in a clinical setting and developing gene delivery shuttles capable of carrying and expressing dystrophin cassettes without toxic or adverse immunologic consequences. We review here recent work from our laboratory to identify sequences within dystrophin that are required to prevent development of dystrophic changes in muscle or which might be able to correct pre-existing damage. We also describe work aimed at developing viral shuttle vectors able to carry and express these dystrophin cassettes at high levels and in a muscle-specific fashion. While great challenges remain in developing methods for systemic gene delivery, we show that a variety of viral vectors are able to carry and express therapeutic levels of dystrophin when delivered directly to mouse skeletal muscle.

Electron microscopic abnormalities of skeletal muscle in patients with collagen VI deficiency in Ullrich's disease.

By electron microscopy, we examined the skeletal muscle from two patients with Ullrich's disease. One patient had a deletion in the collagen VI alpha 2 gene. The muscle biopsy specimens showed no collagen VI immunoreaction, while the expression of collagen IV was increased. Collagen VI is a microfibrillar protein in the extracellular matrix with cell adhesive properties, and collagen IV is a principal component of the basal lamina. Electron microscopy revealed unevenness, extension, and folding of the muscle plasma membrane, and showed thickening and overproduction of the basal lamina. The data show that type VI collagen is certainly one of the important extracellular matrix components maintaining the structural integrity of skeletal muscle, and a defect of the collagen VI protein causes abnormalities of the muscle plasma membrane, dystrophic muscle changes, and up-regulation of collagen IV.

Myosin heavy chain IIa gene mutation E706K is pathogenic and its expression increases with age.

The authors recently described a new autosomal dominant myopathy (OMIM 605637 inclusion body myopathy 3) associated with a missense mutation in the myosin heavy chain (MyHC) IIa gene (MyHC IIa, Human Gene Map [HGM] locus MYH2). Young patients showed minor changes in their muscle biopsies, although dystrophic alterations and rimmed vacuoles with 15- to 20-nm tubulofilaments identical to those in sporadic inclusion body myositis (s-IBM) were observed in some of the adult (especially older) patients. The current study was undertaken to investigate the relation between expression of the mutant MyHC IIa and pathologic changes in muscle. The expression of MyHC IIa in nine muscle specimens from six individuals carrying the mutation was analyzed by immunohistochemistry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a new reverse transcriptase--PCR method to measure the relative abundance of the various MyHC transcripts. Young patients with muscle weakness and minor pathologic changes in muscle expressed MyHC IIa at undetectable levels. MyHC IIa was expressed at high levels in adults with a progressive clinical course and dystrophic muscle changes. In these cases, a large number of muscle fibers were hybrids with expression of more than one MyHC isoform. Both MyHC IIa alleles were equally expressed. The relative level of MyHC IIa transcripts exceeded that of the corresponding protein, indicating an increased turnover of mutated protein. MyHC IIa expression was a consistent finding in muscle fibers with rimmed vacuoles. The clear correlation between pathologic changes and expression of MyHC IIa indicates that defects in MyHC may lead not only to muscle weakness but also to muscle degeneration. The consistent expression of MyHC IIa in muscle fibers with rimmed vacuoles indicates that the breakdown of sarcomeric proteins is a key element in the pathogenesis of rimmed vacuoles of s-IBM type.

Mutations in the fukutin-related protein gene (FKRP) identify limb girdle muscular dystrophy 2I as a milder allelic variant of congenital muscular dystrophy MDC1C

The limb girdle and congenital muscular dystrophies (LGMD and CMD) are characterized by skeletal muscle weakness and dystrophic muscle changes. The onset of symptoms in CMD is within the first few months of life, whereas in LGMD they can occur in late childhood, adolescence or adult life. We have recently demonstrated that the fukutin-related protein gene (FKRP) is mutated in a severe form of CMD (MDC1C), characterized by the inability to walk, leg muscle hypertrophy and a secondary deficiency of laminin alpha2 and alpha-dystroglycan. Both MDC1C and LGMD2I map to an identical region on chromosome 19q13.3. To investigate whether these are allelic disorders, we undertook mutation analysis of FKRP in 25 potential LGMD2I families, including some with a severe and early onset phenotype. Mutations were identified in individuals from 17 families. A variable reduction of alpha-dystroglycan expression was observed in the skeletal muscle biopsy of all individuals studied. In addition, several cases showed a deficiency of laminin alpha2 either by immunocytochemistry or western blotting. Unexpectedly, affected individuals from 15 families had an identical C826A (Leu276Ileu) mutation, including five that were homozygous for this change. Linkage analysis identified at least two possible haplotypes in linkage disequilibrium with this mutation. Patients with the C826A change had the clinically less severe LGMD2I phenotype, suggesting that this is a less disruptive FKRP mutation than those found in MDC1C. The spectrum of LGMD2I phenotypes ranged from infants with an early presentation and a Duchenne-like disease course including cardiomyopathy, to milder phenotypes compatible with a favourable long-term outcome.

Massive muscle cell degeneration in the early stage of merosin-deficient congenital muscular dystrophy.

Primary merosin-deficient congenital muscular dystrophy (CMD) is a severe form of congenital muscular disorder which is caused by mutations in the laminin alpha2 chain gene (LAMA2). The disease is characterized by marked dystrophic changes in skeletal muscles during early infancy, while little is known about the pathological process of the muscle fiber degeneration. Here, we report the immunohistochemical analysis of skeletal muscle in ten patients with primary merosin-deficient CMD using a panel of molecular markers for skeletal muscle proteins, cellular necrosis, and apoptosis. In the youngest patient (a 52 day old baby), prominent massive muscle cell degeneration occurred in association with the deposition of the C5-9 complement membrane attack complex (MAC). Most of the MAC-positive muscle fibers showed a severely deranged immunoreaction to dystrophin, dystroglycans, and other sarcolemmal proteins. In addition, we found scattered positive signals for apoptosis. Similar but milder changes were also observed in six other patients younger than 1 year. In the patients older than 3 years, muscle fibers positive for MAC and apoptotic signals were barely detectable. These findings imply that massive muscle fiber degeneration occurs in the very early stage of merosin-deficient CMD and may contribute to the severe dystrophic changes in muscle from early infancy.