Aclarubicin treatment restores SMN levels to cells derived from type I spinal muscular atrophy patients.

Proximal spinal muscular atrophy (SMA) is a common motor neuron disorder caused by mutation of the telomeric survival of motor neuron gene SMN1. The centromeric survival of motor neuron SMN2 gene is retained in all SMA patients but does not produce sufficient SMN protein to prevent the development of clinical symptoms. The SMN1 and SMN2 genes differ functionally by a single nucleotide change. This change affects the efficiency with which exon 7 is incorporated into the mRNA transcript. Thus, SMN2 produces less full-length mRNA and protein than SMN1. We have screened a library of compounds in order to identify ones that can alter the splicing pattern of the SMN2 gene. Here, we report that the compound aclarubicin increases the retention of exon 7 into the SMN2 transcript. We show that aclarubicin effectively induces incorporation of exon 7 into SMN2 transcripts from the endogenous gene in type I SMA fibroblasts as well as into transcripts from a SMN2 minigene in the motor neuron cell line NSC34. In type I fibroblasts, treatment resulted in an increase in SMN protein and gems to normal levels. Our results suggest that alteration of splicing pattern represents a new approach to modification of gene expression in disease treatment and demonstrate the feasibility of high throughput screens to detect compounds that affect the splicing pattern of a gene.

Use of western immunoblot for evaluation of myocardial dystrophin, alpha-sarcoglycan, and beta-dystroglycan in dogs with idiopathic dilated cardiomyopathy.

OBJECTIVE: To evaluate the potential importance of dystrophin, alpha-sarcoglycan (adhalin), and beta-dystroglycan, by use of western blot analysis, in several breeds of dogs with dilated cardiomyopathy. SAMPLE POPULATION: Myocardial samples obtained from 12 dogs were evaluated, including tissues from 7 dogs affected with dilated cardiomyopathy, 4 control dogs with no identifiable heart disease (positive control), and 1 dog affected with Duchenne muscular dystrophy (negative control for dystrophin). Of the affected dogs, 4 breeds were represented (Doberman Pinscher, Dalmatian, Bullmastiff, and Irish Wolfhound). PROCEDURE: Western blot analysis was used for evaluation of myocardial samples obtained from dogs with and without dilated cardiomyopathy for the presence of dystrophin and 2 of its associated glycoproteins, alpha-sarcoglycan and beta-dystroglycan. RESULTS: Detectable differences were not identified between dogs with and without myocardial disease in any of the proteins evaluated. CONCLUSIONS AND CLINICAL RELEVANCE: Abnormalities in dystrophin, alpha-sarcoglycan, and beta-dystroglycan proteins were not associated with the development of dilated cardiomyopathy in the dogs evaluated in this study. In humans, the development of molecular biological techniques has allowed for the identification of specific causes of dilated cardiomyopathy that were once considered to be idiopathic. The use of similar techniques in veterinary medicine may aid in the identification of the cause of idiopathic dilated cardiomyopathy in dogs, and may offer new avenues for therapeutic intervention.

The survival motor neuron (SMN) protein: effect of exon loss and mutation on protein localization.

Spinal muscular atrophy (SMA) is caused by mutations in the telomeric copy of the survival motor neuron gene (SMN1) but not mutations in the centromeric copy (SMN2). The critical difference between the two genes is a nucleotide difference in exon 7 that affects splicing and causes this exon to be spliced out of most SMN2 transcripts. A majority of the SMN1 gene transcripts contain exon 7. To investigate the effect of exon loss or mutations in SMN on protein localization, 15 SMN constructs were prepared and transfected into COS-7 cells and fibroblasts derived from a type I SMA patient. Loss of exon 5 (Iso5-SMN), a putative nuclear localization signal in exon 2, and the G279V point mutation had little effect on SMN localization. Loss of both exons 5 and 7 (Iso57-SMN) resulted in low gem numbers and the localization of the majority of the SMN protein to the cytoplasm. Cells expressing constructs lacking only exon 7 (Iso7-SMN) did not produce large numbers of gems in general, although there were a few cells that had a staining pattern similar to cells transfected with a full-length (Full-SMN) construct. HeLa cells stably transfected with full-length SMN or Iso7-SMN did not overexpress SMN, and both constructs produced a similar localization of the protein, although Iso7-SMN formed gems less efficiently. Removal of the amino-terminus, deletion of the conserved domain in exon 2A, and the mutation Y272C all caused accumulation of SMN in the nucleus, sometimes in large aggregates. These findings suggest that the amino-terminal domain of SMN is essential for the correct cellular distribution of SMN, whereas Iso7-SMN is capable of forming gems, albeit at a reduced efficiency.

Animal models of spinal muscular atrophy.

Proximal spinal muscular atrophy (SMA) is the second most common autosomal recessive inherited disorder in humans. It is the most common genetic cause of infant mortality. As yet, there is no cure for this neuromuscular disorder which affects the lower motor neurons and proximal muscles of the limbs and trunk. In the last decade, significant advances have been made in understanding this disease, from linkage analysis to isolating the defective gene and identifying its protein product. This review summarizes the most recent advance in SMA research: the development of animal models of the disease, in particular mouse models of SMA. The SMA mice that we describe here present with symptoms similar to those seen in SMA patients. They promise to further the understanding of the molecular basis of this disease and demonstrate the feasibility of using the intact SMN2 gene, found in all SMA patients, as a means of treating this disorder.

The human centromeric survival motor neuron gene (SMN2) rescues embryonic lethality in Smn(-/-) mice and results in a mouse with spinal muscular atrophy.

Proximal spinal muscular atrophy (SMA) is a common motor neuron disease in humans and in its most severe form causes death by the age of 2 years. It is caused by defects in the telomeric survival motor neuron gene ( SMN1 ), but patients retain at least one copy of a highly homologous gene, centromeric SMN ( SMN2 ). Mice possess only one survival motor neuron gene ( Smn ) whose loss is embryonic lethal. Therefore, to obtain a mouse model of SMA we created transgenic mice that express human SMN2 and mated these onto the null Smn (-/-)background. We show that Smn (-/-); SMN2 mice carrying one or two copies of the transgene have normal numbers of motor neurons at birth, but vastly reduced numbers by postnatal day 5, and subsequently die. This closely resembles a severe type I SMA phenotype in humans and is the first report of an animal model of the disease. Eight copies of the transgene rescues this phenotype in the mice indicating that phenotypic severity can be modulated by SMN2 copy number. These results show that SMA is caused by insufficient SMN production by the SMN2 gene and that increased expression of the SMN2 gene may provide a strategy for treating SMA patients.

The survival motor neuron protein in spinal muscular atrophy.

The 38 kDa survival motor neuron (SMN) protein is encoded by two ubiquitously expressed genes: telomeric SMN (SMN(T)) and centromeric SMN (SMN(C)). Mutations in SMN(T), but not SMN(C), cause proximal spinal muscular atrophy (SMA), an autosomal recessive disorder that results in loss of motor neurons. SMN is found in the cytoplasm and nucleus. The nuclear form is located in structures termed gems. Using a panel of anti-SMN antibodies, we demonstrate that the SMN protein is expressed from both the SMN(T) and SMN(C) genes. Western blot analysis of fibroblasts from SMA patients with various clinical severities of SMA showed a moderate reduction in the amount of SMN protein, particularly in type I (most severe) patients. Immunocytochemical analysis of SMA patient fibroblasts indicates a significant reduction in the number of gems in type I SMA patients and a correlation of the number of gems with clinical severity. This correlation to phenotype using primary fibroblasts may serve as a useful diagnostic tool in an easily accessible tissue. SMN is expressed at high levels in brain, kidney and liver, moderate levels in skeletal and cardiac muscle, and low levels in fibroblasts and lymphocytes. In SMA patients, the SMN level was moderately reduced in muscle and lymphoblasts. In contrast, SMN was expressed at high levels in spinal cord from normals and non-SMA disease controls, but was reduced 100-fold in spinal cord from type I patients. The marked reduction of SMN in type I SMA spinal cords is consistent with the features of this motor neuron disease. We suggest that disruption of SMN(T) in type I patients results in loss of SMN from motor neurons, resulting in the degeneration of these neurons.

Gene therapy for muscle diseases.

Duchenne muscular dystrophy involves progressive degeneration of the skeletal and cardiac muscles, resulting in premature death. A number of methods are currently being developed for the treatment of Duchenne muscular dystrophy and other neuromuscular disorders. A number of the viral vector systems, myoblast transfer, and direct injection techniques that are currently under investigation for the treatment of neuromuscular disorders are reviewed here.

Characterization of translational frame exception patients in Duchenne/Becker muscular dystrophy.

The clinical progression of Duchenne muscular dystrophy (DMD) patients with deletions can be predicted in 93% of cases by whether the deletion maintains or disrupts the translational reading frame (frameshift hypothesis). We have identified and studied a number of patients who have deletions that do not conform to the translational frame hypothesis. The most common exception to the frameshift hypothesis is the deletion of exons 3 to 7 which disrupts the translational reading frame. We identified a Becker muscular dystrophy (BMD) patient, an intermediate, and a DMD patient with this deletion. In all three cases, dystrophin was detected and localized to the membrane. One DMD patient with an inframe deletion of exons 4-18 produced no dystrophin. One patient with a mild intermediate phenotype and a deletion of exon 45, which shifts the reading frame, produced no dystrophin. Two patients with large inframe deletions had discordant phenotypes (exons 3-41, DMD; exons 13-48, BMD), but both produced dystrophin that localized to the sarcolemma. The DMD patient, 113, indicates that dystrophin with an intact carboxy terminus can be produced in Duchenne patients at levels equivalent to some Beckers. The dystrophin analysis from these patients, together with patients reported in the literature, indicate that more than one domain can localize dystrophin to the sarcolemma. Lastly, the data shows that although most patients show correlation of clinical severity to molecular data, there are rare patients which do not conform.

Somatic reversion/suppression in Duchenne muscular dystrophy (DMD): evidence supporting a frame-restoring mechanism in rare dystrophin-positive fibers.

Many Duchenne muscular dystrophy (DMD) patients are known to have rare staining dystrophin-positive fibers, termed "revertants." The precise etiology of these rare fibers is unknown. The most likely explanation, however, is somatic mosaicism or somatic reversion/suppression. Immunocytochemistry was performed on serial sections from deleted and nondeleted patients, with a panel of antibodies--9219, 1377, 9218, and Dys-2--that span dystrophin. Both familial and nonfamilial patients possessed revertants. Either the same clusters or individual revertant fibers stained with amino- and carboxyl-terminal antibodies in all 14 DMD patients. In patients with deletions, revertants did not stain with antibodies raised to polypeptide sequences within the deletion. These results indicate that positively staining fibers are not the result of somatic mosaicism in deleted patients. Five of 10 patients without deletions had revertant fibers. In two of these patients, the revertant fibers did not stain with antibody 9218, which was generated against amino acids 2305-2554 and which corresponds to exons 48-52. The remaining antibodies from the panel stained the same fibers on separate serial sections in these two patients. The most likely mechanism giving rise to these positively staining fibers is a second site in-frame deletion. Antibodies generated to polypeptide sequences within deletions can be used to control for the natural occurrence of revertant fibers in myoblast transfer studies and may be useful in the detection of point mutations.

Dystrophin expression and somatic reversion in prednisone-treated and untreated Duchenne dystrophy. CIDD Study Group.

The mechanism by which prednisone improves muscle strength and function in Duchenne muscular dystrophy (DMD) is unknown. We addressed the possibility that clinical improvement was related to prednisone-induced alterations in skeletal muscle dystrophin. We performed muscle biopsies on patients at the conclusion of a randomized, double-blind, 6-month trial of prednisone and analyzed dystrophin content using Western blots and antibody staining of tissue sections. These studies demonstrated no significant differences in dystrophin content between treatment (prednisone 1.5 mg/kg/d, n = 12; prednisone 0.75 mg/kg/d, n = 9) and placebo (n = 12) groups. Of interest, however, was the presence of varying numbers of dystrophin-positive fibers (revertants) occurring individually or in clusters in antibody-stained tissue sections of more than one-half of the Duchenne patients. Mutation analysis revealed that revertants occurred in DMD patients with identifiable deletions half of the Duchenne patients. Mutation analysis revealed that revertants occurred in DMD patients with identifiable deletions or duplications, and in nondeletion patients. Prednisone treatment did not influence the prevalence of revertants. Revertants are most likely due to a second-site mutation occurring in a somatic cell allowing for restoration of the translational reading frame of the dystrophin transcript.