Laminin alpha1 chain reduces muscular dystrophy in laminin alpha2 chain deficient mice

Laminin (LN) alpha2 chain deficiency in humans and mice leads to severe forms of congenital muscular dystrophy (CMD). Here, we investigated whether LNalpha1 chain in mice can compensate for the absence of LNalpha2 chain and prevent the development of muscular dystrophy. We generated mice expressing a LNalpha1 chain transgene in skeletal muscle of LNalpha2 chain deficient mice. LNalpha1 is not normally expressed in muscle, but the transgenically produced LNalpha1 chain was incorporated into muscle basement membranes, and normalized the compensatory changes of expression of certain other laminin chains (alpha4, beta2). In 4-month-old mice, LNalpha1 chain could fully prevent the development of muscular dystrophy in several muscles, and partially in others. The LNalpha1 chain transgene not only reversed the appearance of histopathological features of the disease to a remarkable degree, but also greatly improved health and longevity of the mice. Correction of LNalpha2 chain deficiency by LNalpha1 chain may serve as a paradigm for gene therapy of CMD in patients.     

Functional requirements for fukutin-related protein in the Golgi apparatus

Two forms of congenital muscular dystrophy (CMD), Fukuyama CMD and CMD type 1C (MDC1C) are caused by mutations in the genes encoding two putative glycosyltransferases, fukutin and fukutin-related protein (FKRP). Additionally, mutations in the FKRP gene also cause limb-girdle muscular dystrophy type 2I (LGMD2I), a considerably milder allelic variant than MDC1C. All of these diseases are associated with secondary changes in muscle alpha-dystroglycan expression. To elucidate the function of FKRP and fukutin and examine the effects of MDC1C patient mutations, we have determined the mechanism for the subcellular location of each protein. FKRP and fukutin are targeted to the medial-Golgi apparatus through their N-termini and transmembrane domains. Overexpression of FKRP in CHO cells alters the post-translational processing of alpha- and beta-dystroglycan inhibiting maturation of the two isoforms. Mutations in the DxD motif in the putative active site of the protein or in the Golgi-targeting sequence, which cause FKRP to be inefficiently trafficked to the Golgi apparatus, did not alter dystroglycan processing in vitro. The P448L mutation in FKRP that causes congenital muscular dystrophy changes a conserved amino acid resulting in the mislocalization of the mutant protein in the cell that is unable to alter dystroglycan processing. Our data show that FKRP and fukutin are Golgi-resident proteins and that FKRP is required for the post-translational modification of dystroglycan. Aberrant processing of dystroglycan caused by a mislocalized FKRP mutant could be a novel mechanism that causes congenital muscular dystrophy.     

The expanding phenotype of laminin alpha2 chain (merosin) abnormalities: case series and review

Initial reports of patients with laminin alpha2 chain (merosin) deficiency had a relatively homogeneous phenotype, with classical congenital muscular dystrophy (CMD) characterised by severe muscle weakness, inability to achieve independent ambulation, markedly raised creatine kinase, and characteristic white matter hypodensity on cerebral magnetic resonance imaging. We report a series of five patients with laminin alpha2 deficiency, only one of whom has this severe classical CMD phenotype, and review published reports to characterise the expanded phenotype of laminin alpha2 deficiency, as illustrated by this case series. While classical congenital muscular dystrophy with white matter abnormality is the commonest phenotype associated with laminin alpha2 deficiency, 12% of reported cases have later onset, slowly progressive weakness more accurately designated limb-girdle muscular dystrophy. In addition, the following clinical features are reported with increased frequency: mental retardation (~6%), seizures (~8%), subclinical cardiac involvement (3-35%), and neuronal migration defects (4%). At least 25% of patients achieve independent ambulation. Notably, three patients with laminin alpha2 deficiency were asymptomatic, 10 patients had normal MRI (four with LAMA2 mutations reported), and between 10-20% of cases had maximum recorded creatine kinase of less than 1000 U/l. LAMA2 mutations have been identified in 25% of cases. Sixty eight percent of these have the classical congenital muscular dystrophy, but this figure is likely to be affected by ascertainment bias. We conclude that all dystrophic muscle biopsies, regardless of clinical phenotype, should be studied with antibodies to laminin alpha2. In addition, the use of multiple antibodies to different regions of laminin alpha2 may increase the diagnostic yield and provide some correlation with severity of clinical phenotype.     

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.     

Clinical and molecular study in congenital muscular dystrophy with partial laminin alpha 2 (LAMA2) deficiency

Complete laminin alpha2 (LAMA2) deficiency causes approximately half of congenital muscular dystrophy (CMD) cases. Many loss-of-function mutations have been reported in these severe, neonatal-onset patients, but only single missense mutations have been found in milder CMD with partial laminin alpha2 deficiency. Here, we studied nine patients diagnosed with CMD who showed abnormal white-matter signal at brain MRI and partial deficiency of laminin alpha2 on immunofluorescence of muscle biopsy. We screened the entire 9.5 kb laminin alpha2 mRNA from patient muscle biopsy by direct capillary automated sequencing, single strand conformational polymorphism (SSCP), or denaturing high performance liquid chromatography (DHPLC) of overlapping RT-PCR products followed by direct sequencing of heteroduplexes. We identified laminin alpha2 sequence changes in six of nine CMD patients. Each of the gene changes identified, except one, was novel, including three missense changes and two splice-site mutations. The finding of partial laminin alpha2 deficiency by immunostaining is not specific for laminin alpha2 gene mutation carriers, with only two patients (22%) showing clear causative mutations, and an additional three patients (33%) showing possible mutations. The clinical presentation and disease progression was homogeneous in the laminin alpha2-mutation positive and negative CMD patients.     

Platelet-derived growth factor and its receptors are related to the progression of human muscular dystrophy: an immunohistochemical study

This study has examined the immunological localization of platelet-derived growth factor (PDGF)-A, PDGF-B, and PDGF receptor (PDGFR) alpha and beta to clarify their role in the progression of muscular dystrophy. Biopsied frozen muscles from patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and congenital muscular dystrophy (CMD) were analysed immunohistochemically using antibodies raised against PDGF-A, PDGF-B, and PDGFR alpha and beta. Muscles from two dystrophic mouse models (dy and mdx mice) were also immunostained with antibodies raised against PDGFR alpha and beta. In normal human control muscle, neuromuscular junctions and vessels were positively stained with antibodies against PDGF-A, PDGF-B, PDGFR alpha and PDGFR beta. In human dystrophic muscles, PDGF-A, PDGF-B, PDGFR alpha and PDGFR beta were strongly immunolocalized in regenerating muscle fibres and infiltrating macrophages. PDGFR alpha was also immunolocalized to the muscle fibre sarcolemma and necrotic fibres. The most significant finding in this study was a remarkable overexpression of PDGFR beta and, to a lesser extent, PDGFR alpha in the endomysium of DMD and CMD muscles. PDGFR was also overexpressed in the interstitium of muscles from dystrophic mice, particularly dy mice. Double immunolabelling revealed that activated interstitial fibroblasts were clearly positive for PDGFR alpha and beta. However, DMD and CMD muscles with advanced fibrosis showed very poor reactivity against PDGF and PDGFR. Those findings were confirmed by immunoblotting with PDGFR beta. These findings indicate that PDGF and its receptors are significantly involved in the active stage of tissue destruction and are associated with the initiation or promotion of muscle fibrosis. They also have roles in muscle fibre regeneration and signalling at neuromuscular junctions in both normal and diseased muscle.     

Merosin (laminin-2) localization in basal lamina of normal skeletal muscle fibers and changes in plasma membrane of merosin-deficient skeletal muscle fibers

Primary deficiency of merosin causes a severe congenital muscular dystrophy (CMD) and a mouse dystrophy (dy/dy mouse). Also, its secondary deficiency is seen in some CMD with abnormal glycosylation of -dystroglycan, an extracellular membrane protein, which is the receptor of merosin and binds to dystrophin underlying the sarcolenma via -dystroglycan, a transmembrane protein. In immunogold and freeze-etch electron microscopic studies, merosin in basal lamina of normal skeletal muscles has a zonation in the distribution and is localized at the lamina lucida of muscle basal lamina, and dystrophin molecules are often closed to merosin molecules at the inside and outside surface of muscle plasma membrane. Moreover, merosin molecules exist as the short fine cross-bridge fibrils connecting the basal lamina to the neighboring outer leaflet of the muscle plasma membrane. In freeze-fracture studies, the changes in muscle plasma membranes of dy/dy mice reveal a markedly decreased density of orthogonal arrays (OAs) but normal density of intramembranous particles (IMPs), whereas depletions of IMPs with decreased OAs have been found in Fukyama-type congenital muscular dystrophy, Duchenne muscular dystrophy, and mdx mice. Thus, further studies including the functional role of OAs would be required to understand the pathomechanism of merosin-deficient CMD.     

Integrin alpha 7 beta 1 in muscular dystrophy/myopathy of unknown etiology

To investigate the role of integrin alpha 7 in muscle pathology, we used a "candidate gene" approach in a large cohort of muscular dystrophy/myopathy patients. Antibodies against the intracellular domain of the integrin alpha 7A and alpha 7B were used to stain muscle biopsies from 210 patients with muscular dystrophy/myopathy of unknown etiology. Levels of alpha 7A and alpha 7B integrin were found to be decreased in 35 of 210 patients (approximately 17%). In six of these patients no integrin alpha 7B was detected. Screening for alpha 7B mutation in 30 of 35 patients detected only one integrin alpha 7 missense mutation (the mutation on the second allele was not found) in a patient presenting with a congenital muscular dystrophy-like phenotype. No integrin alpha 7 gene mutations were identified in all of the other patients showing integrin alpha 7 deficiency. In the process of mutation analysis, we identified a novel integrin alpha 7 isoform presenting 72-bp deletion. This isoform results from a partial deletion of exon 21 due to the use of a cryptic splice site generated by a G to A missense mutation at nucleotide position 2644 in integrin alpha 7 cDNA. This spliced isoform is present in about 12% of the chromosomes studied. We conclude that secondary integrin alpha 7 deficiency is rather common in muscular dystrophy/myopathy of unknown etiology, emphasizing the multiple mechanisms that may modulate integrin function and stability.     

Genotype/phenotype analysis in Chinese laminin‐α2 deficient congenital muscular dystrophy patients

Laminin‐α2 deficient congenital muscular dystrophy (CMD) is an autosomal recessive disorder characterized by severe muscular dystrophy, which is typically associated with abnormal white matter. In this study, we assessed 43 CMD patients with typical white matter abnormality and laminin‐α2 deficiency (complete or partial) diagnosed by immunohistochemistry to determine the clinical and molecular genetic characteristics of laminin‐α2 deficient CMD. LAMA2 gene mutation analysis was performed by direct sequencing of genomic DNAs. Exonic deletion or duplication was identified by multiplex ligation‐dependent probe amplification (MLPA) and verified by high‐density oligonucleotide‐based CGH microarrays. Gene mutation analysis revealed 86 LAMA2 mutations (100%); 15 known and 37 novel. Among these mutations, 73.9% were nonsense, splice‐site or frameshift and 18.8% were deletions of one or more exons. Genetic characterization of affected families will be valuable in prenatal diagnosis of CMD in the Chinese population.     

Fukutin is required for maintenance of muscle integrity,cortical histiogenesis and normal eye development

Fukuyama-type congenital muscular dystrophy (FCMD), one of the most common autosomal-recessive disorders in Japan, is characterized by congenital muscular dystrophy associated with brain malformation due to a defect during neuronal migration. Through positional cloning, we previously identified the gene for FCMD, which encodes the fukutin protein. Here we report that chimeric mice generated using embryonic stem cells targeted for both fukutin alleles develop severe muscular dystrophy, with the selective deficiency of alpha-dystroglycan and its laminin-binding activity. In addition, these mice showed laminar disorganization of the cortical structures in the brain with impaired laminin assembly, focal interhemispheric fusion, and hippocampal and cerebellar dysgenesis. Further, chimeric mice showed anomaly of the lens, loss of laminar structure in the retina, and retinal detachment. These results indicate that fukutin is necessary for the maintenance of muscle integrity, cortical histiogenesis, and normal ocular development and suggest the functional linkage between fukutin and alpha-dystroglycan.     

Severe classical congenital muscular dystrophy and merosin expression

Vajsar J, Chitayat D, Becker LE, Ho M, Ben-Zeev B, Jay V. Severe classical congenital muscular dystrophy and merosin expression. Clin Genet 1998: 54: 193–198. 0 Munksgaard, 1998
It has been suggested that patients with autosomal recessive merosin deficient congenital muscular dystrophy (CMD), as opposed to the merosin positive cases form a homogeneous subgroup of a clinically more severe form of CMD. We examined merosin expression in muscle biopsies from five children with the severe classical form of CMD. Merosin deficiency was found only in 1 patient, a 6–year-old female, with abnormal brain myelination. However, her initial biopsy did not reveal the classical picture of dystrophy. The four merosin positive cases exhibited severe muscle weakness but their brain imagings were normal. There were no familial cases, except for the mother of 1 patient who had a milder form of the disease, suggesting an autosomal dominant mode of inheritance.
In contrast to previous reports, the merosin deficient CMD cases were rare in our group. Furthermore, merosin positive cases were also associated with severe phenotype suggesting that a severe phenotype is not exclusive to merosin deficient cases. Finally, the absence of merosin in a neonate with hypotonia and weakness can be helpful in making a definitive diagnosis of CMD, even though the dystrophic process may not be evident yet and histology may be non-specific.     

Localization and functional analysis of the LARGE family of glycosyltransferases: significance for muscular dystrophy

The dystroglycanopathies are a novel group of human muscular dystrophies due to mutations in known or putative glycosyltransferase enzymes. They share the common pathological feature of a hypoglycosylated form of alpha-dystroglycan, diminishing its ability to bind extracellular matrix ligands. The LARGE glycosyltransferase is mutated in both the myodystrophy mouse and congenital muscular dystrophy type 1D (MDC1D). We have transfected various cell lines with a variety of LARGE expression constructs in order to characterize their subcellular localization and effect on alpha-dystroglycan glycosylation. Wild-type LARGE co-localized with the Golgi marker GM130 and stimulated the production of highly glycosylated alpha-dystroglycan (hyperglycosylation). MDC1D mutants had no effect on alpha-dystroglycan glycosylation and failed to localize correctly, confirming their pathogenicity. The two predicted catalytic domains of LARGE contain three conserved DxD motifs. Systematically mutating each of these motifs to NNN resulted in the mislocalization of one construct, while all failed to have any effect on alpha-dystroglycan glycosylation. A construct lacking the transmembrane domain also failed to localize at the Golgi apparatus. These results indicate that LARGE needs to both physically interact with alpha-dystroglycan and function as a glycosyltransferase in order to stimulate alpha-dystroglycan hyperglycosylation. We have also cloned and overexpressed a homologue of LARGE, glycosyltransferase-like 1B (GYLTL1B). Like LARGE it localized to the Golgi apparatus and stimulated alpha-dystroglycan hyperglycosylation. These results suggest that GYLTL1B may be a candidate gene for muscular dystrophy and that its overexpression could compensate for the deficiency of both LARGE and other glycosyltransferases.     

Dissociation of the dystroglycan complex in caveolin-3-deficient limb girdle muscular dystrophy

Limb girdle muscular dystrophy is a group of clinically and genetically heterogeneous disorders inherited in an autosomal recessive or dominant mode. Caveolin-3, the muscle-specific member of the caveolin gene family, is implicated in the pathogenesis of autosomal dominant limb girdle muscular dystrophy 1C. Here we report on a 4-year-old girl presenting with myalgia and muscle cramps due to a caveolin-3 deficiency in her dystrophic skeletal muscle as a result of a heterozygous 136G-->A substitution in the caveolin-3 gene. The novel sporadic missense mutation in the caveolin signature sequence of the caveolin-3 gene changes an alanine to a threonine (A46T) and prevents the localization of caveolin-3 to the plasma membrane in a dominant negative fashion. Caveolin-3 has been suggested to interact with the dystrophin-glycoprotein complex, which in striated muscle fibers links the cytoskeleton to the extracellular matrix and with neuronal nitric oxide synthase. Similar to dystrophin-deficient Duchenne muscular dystrophy, a secondary decrease in neuronal nitric oxide synthase and alpha-dystroglycan expression was detected in the caveolin-3-deficient patient. These results implicate an important function of the caveolin signature sequence and common mechanisms in the pathogenesis of dystrophin-glycoprotein complex-associated muscular dystrophies with caveolin-3-deficient limb girdle muscular dystrophy.     

Refinement of the laminin alpha2 chain locus to human chromosome 6q2 in severe and mild merosin deficient congenital muscular dystrophy.

About half of the children with classical congenital muscular dystrophy (CMD) show an absence in their skeletal muscle of laminin alpha2 chain, one of the components of the extracellular matrix protein, merosin. Linkage analysis implicated the laminin alpha2 chain gene (LAMA2) on chromosome 6q2, now confirmed by the discovery of mutations in the laminin alpha2 chain gene. We have further investigated the location of the LAMA2 locus on chromosome 6q2, using both linkage analysis in nine informative families and homozygosity mapping in 13 consanguineous families. Four of these families only had mild or moderate down regulation of laminin alpha2 chain expression and a milder phenotype; the rest had no protein or only a trace. Haplotype analysis in all the informative families, including those with partial laminin alpha2 expression, was compatible with linkage to chromosome 6q2. This observation expands the spectrum of the phenotype secondary to laminin alpha2 chain deficiency. Our results suggest that the LAMA2 locus is more centromeric than previously proposed. Recombinant events place the locus between markers D6S470 and D6S1620 in an interval of less than 3 cM.     

Abnormalities in alpha-dystroglycan expression in MDC1C and LGMD2I muscular dystrophies

We recently identified mutations in the fukutin related protein (FKRP) gene in patients with congenital muscular dystrophy type 1C (MDC1C) and limb girdle muscular dystrophy type 2I (LGMD2I). The sarcolemma of these patients typically displays an immunocytochemical reduction of alpha-dystroglycan. In this report we extend these observations and report a clear correlation between the residual expression of alpha-dystroglycan and the phenotype. Three broad categories were identified. Patients at the severe end of the clinical spectrum (MDC1C) were compound heterozygote between a null allele and a missense mutation or carried two missense mutations and displayed a profound depletion of alpha-dystroglycan. Patients with LGMD with a Duchenne-like severity typically had a moderate reduction in alpha-dystroglycan and were compound heterozygotes between a common C826A (Leu276Ileu) FKRP mutation and either a missense or a nonsense mutation. Individuals with the milder form of LGMD2I were almost invariably homozygous for the Leu276Ile FKRP mutation and showed a variable but subtle alteration in alpha-dystroglycan immunolabeling. Our data therefore suggest a correlation between a reduction in alpha-dystroglycan, the mutation and the clinical phenotype in MDC1C and LGMD2I which supports the hypothesis that dystroglycan plays a central role in the pathogenesis of these disorders.     

Severe congenital muscular dystrophy in a Mexican family with a new nonsense mutation (R2578X) in the laminin alpha-2 gene

The congenital muscular dystrophies (CMDs) are a heterogeneous group of autosomal recessive disorders. Approximately one half of cases diagnosed with classic CMD show primary deficiency of the laminin alpha2 chain of merosin. Complete absence of this protein is usually associated with a severe phenotype characterized by drastic muscle weakness and characteristic changes in white matter in cerebral magnetic resonance imaging (MRI). Here we report an 8-month-old Mexican female infant, from a consanguineous family, with classical CMD. Serum creatine kinase was elevated, muscle biopsy showed dystrophic changes, and there were abnormalities in brain MRI. Immunofluorescence analysis demonstrated the complete absence of laminin alpha2. In contrast, expression of alpha-, beta-, gamma-, and delta-sarcoglycans and dystrophin, all components of the dystrophin-glycoprotein complex, appeared normal. A homozygous C long right arrow T substitution at position 7781 that generated a stop codon in the G domain of the protein was identified by mutation analysis of the laminin alpha2 gene ( LAMA2). Sequence analysis on available DNA samples of the family showed that parents and other relatives were carriers of the mutation.     

Biochemical and ultrastructural evidence of endoplasmic reticulum stress in LGMD2I

Limb girdle muscular dystrophy type 2I (LGMD2I) is due to mutations in the fukutin-related protein gene (FKRP), encoding a putative glycosyltransferase involved in alpha-dystroglycan processing. To further characterize the molecular pathogenesis of LGMD2I, we conducted a histological, immunohistochemical, ultrastructural and molecular analysis of ten muscle biopsies from patients with molecularly diagnosed LGMD2I. Hypoglycosylation of alpha-dystroglycan was observed in all FKRP-mutated patients. Muscle histopathology was consistent with either severe muscular dystrophy or myopathy with a mild inflammatory response consisting of up-regulation of class I major histocompatibility complex in skeletal muscle fibers and small foci of mononuclear cells. At the ultrastructural level, muscle fibers showed focal thinning of basal lamina and swollen endoplasmic reticulum cisternae with membrane re-arrangement. The pathways of the unfolded protein response (UPR; glucose-regulated protein 78 and CHOP) were significantly activated in LGMD2I muscle tissue. Our data suggest that the UPR response is activated in LGMD2I muscle biopsies, and the observed histopathological and ultrastructural alterations may be related to sarcoplasmic structures involved in FKRP and alpha-dystroglycan metabolism and malfunctioning.     

Elimination of myostatin does not combat muscular dystrophy in dy mice but increases postnatal lethality

Myostatin is a TGF-beta family member and a negative regulator of skeletal muscle growth. It has been proposed that reduction or elimination of myostatin could be a treatment for degenerative muscle diseases such as muscular dystrophy. Laminin-deficient congenital muscular dystrophy is one of the most severe forms of muscular dystrophy. To test the possibility of ameliorating the dystrophic phenotype in laminin deficiency by eliminating myostatin, we crossed dy(W) laminin alpha2-deficient and myostatin null mice. The resulting double-deficient dy(W)/dy(W);Mstn(-/-) mice had a severe clinical phenotype similar to that of dy(W)/dy(W) mice, even though muscle regeneration was increased. Degeneration and inflammation of muscle were not alleviated. The pre-weaning mortality of dy(W)/dy(W);Mstn(-/-) mice was increased compared to dy(W)/dy(W), most likely due to significantly less brown and white fat in the absence of myostatin, and postweaning mortality was not significantly improved. These results show that eliminating myostatin in laminin-deficiency promotes muscle formation, but at the expense of fat formation, and does not reduce muscle pathology. Any future therapy based on myostatin may have undesirable side effects.