Rumaisa Bashir

Dysferlin deletion in SJL mice (SJL-Dysf) defines a natural model for limb girdle muscular dystrophy 2B.

Dysferlin deletion in SJL mice (SJL- Dysf ) defines a natural model for limb girdle muscular dystrophy 2B

Recessive Mutations in the Putative Calcium-Activated Chloride Channel Anoctamin 5 Cause Proximal LGMD2L and Distal MMD3 Muscular Dystrophies

The recently described human anion channel Anoctamin (ANO) protein family comprises at least ten members, many of which have been shown to correspond to calcium-activated chloride channels. To date, the only reported human mutations in this family of genes are dominant mutations in ANO5 (TMEM16E, GDD1) in the rare skeletal disorder gnathodiaphyseal dysplasia. We have identified recessive mutations in ANO5 that result in a proximal limb-girdle muscular dystrophy (LGMD2L) in three French Canadian families and in a distal non-dysferlin Miyoshi myopathy (MMD3) in Dutch and Finnish families. These mutations consist of a splice site, one base pair duplication shared by French Canadian and Dutch cases, and two missense mutations. The splice site and the duplication mutations introduce premature-termination codons and consequently trigger nonsense-mediated mRNA decay, suggesting an underlining loss-of-function mechanism. The LGMD2L phenotype is characterized by proximal weakness, with prominent asymmetrical quadriceps femoris and biceps brachii atrophy. The MMD3 phenotype is associated with distal weakness, of calf muscles in particular. With the use of electron microscopy, multifocal sarcolemmal lesions were observed in both phenotypes. The phenotypic heterogeneity associated with ANO5 mutations is reminiscent of that observed with Dysferlin (DYSF) mutations that can cause both LGMD2B and Miyoshi myopathy (MMD1). In one MMD3-affected individual, defective membrane repair was documented on fibroblasts by membrane-resealing ability assays, as observed in dysferlinopathies. Though the function of the ANO5 protein is still unknown, its putative calcium-activated chloride channel function may lead to important insights into the role of deficient skeletal muscle membrane repair in muscular dystrophies.

Autosomal dominant retinitis pigmentosa: four new mutations in rhodopsin, one of them in the retinal attachment site

Several mutations in the rhodopsin gene in patients affected by autosomal dominant retinitis pigmentosa (ADRP) have recently been described. We report four new rhodopsin mutations in ADRP families, initially identified as hetero-duplexed PCR fragments on hydrolink gels. One is an in-frame 12-bp deletion of codons 68 to 71. The other three are point mutations involving codons 190, 211, and 296. Each alters the amino acid encoded. The codon 190 mutation has been detected in 2 from a panel of 34 ADRP families, while the remaining mutations were seen in single families. This suggests that, consistent with a dominant condition, no single mutation will account for a large fraction of ADRP cases. The base substitution in codon 296 alters the lysine residue that functions as the attachment site for 11-cis-retinal, mutating it to glutamic acid. This mutation occurs in a family with an unusually severe phenotype, resulting in early onset of disease and cataracts in the third or fourth decade of life. This result demonstrates a correlation between the location of the mutation and the severity of phenotype in rhodopsin RP.

Secondary reduction in calpain 3 expression in patients with limb girdle muscular dystrophy type 2B and Miyoshi myopathy (primary dysferlinopathies)

Dysferlin is the protein product of the gene (DYSF) that is defective in patients with limb girdle muscular dystrophy type 2B and Miyoshi myopathy. Calpain 3 is the muscle-specific member of the calcium activated neutral protease family and primary mutations in the CAPN3 gene cause limb girdle muscular dystrophy type 2A. The functions of both proteins remain speculative. Here we report a secondary reduction in calpain 3 expression in eight out of 16 patients with a primary dysferlinopathy and clinical features characteristic of limb girdle muscular dystrophy type 2B or Miyoshi myopathy. Previously CAPN3 analysis had been undertaken in three of these patients and two showed seemingly innocuous missense mutations, changing calpain 3 amino acids to those present in the sequences of calpains 1 and 2. These results suggest that there may be an association between dysferlin and calpain 3, and further analysis of both genes may elucidate a novel functional interaction. In addition, an association was found between prominent expression of smaller forms of the 80 kDa fragment of laminin alpha 2 chain (merosin) and dysferlin-deficiency.

Mutation analysis of the spastin gene (SPG4) in patients with hereditary spastic paraparesis

BACKGROUND Hereditary spastic paraparesis is a genetically heterogeneous condition. Recently, mutations in the spastin gene were reported in families linked to the common SPG4 locus on chromosome 2p21-22. OBJECTIVES To study a population of patients with hereditary spastic paraparesis for mutations in the spastin gene (SPG4) on chromosome 2p21-22. METHODS DNA from 32 patients (12 from families known to be linked to SPG4) was analysed for mutations in the spastin gene by single strand conformational polymorphism analysis and sequencing. All patients were also examined clinically. RESULTS ThirteenSPG4 mutations were identified, 11 of which are novel. These mutations include missense, nonsense, frameshift, and splice site mutations, the majority of which affect the AAA cassette. We also describe a nucleotide substitution outside this conserved region which appears to behave as a recessive mutation. CONCLUSIONS Recurrent mutations in the spastin gene are uncommon. This reduces the ease of mutation detection as a part of the diagnostic work up of patients with hereditary spastic paraparesis. Our findings have important implications for the presumed function of spastin and schemes for mutation detection in HSP patients.

Genomic organization of the dysferlin gene and novel mutations in Miyoshi myopathy

Objective: Mutations in the skeletal muscle gene dysferlin cause two autosomal recessive forms of muscular dystrophy: Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B). The purpose of this study was to define the genomic organization of the dysferlin gene and conduct mutational screening and a survey of clinical features in 21 patients with defined molecular defects in the dysferlin gene. Methods: Genomic organization of the gene was determined by comparing the dysferlin cDNA and genomic sequence in P1-derived artificial chromosomes (PACs) containing the gene. Mutational screening entailed conformational analysis and sequencing of genomic DNA and cDNA. Clinical records of patients with defined dysferlin gene defects were reviewed retrospectively. Results: The dysferlin gene encompasses 55 exons spanning over 150 kb of genomic DNA. Mutational screening revealed nine novel mutations associated with MM. The range of onset in this patient group was narrow with a mean of 19.0 ± 3.9 years. Conclusion: This study confirms that the dysferlin gene is mutated in MM and LGMD2B and extends understanding of the timing of onset of the disease. Knowledge of the genomic organization of the gene will facilitate mutation detection and investigations of the molecular biologic properties of the dysferlin gene.

Clinical and pathologic findings in hereditary spastic paraparesis with spastin mutation

Objective To describe a family with chromosome 2p‐linked hereditary spastic paraparesis (HSP) associated with dementia and illustrate the cerebral pathology associated with this disorder. Background HSP comprises a heterogeneous group of inherited disorders in which the main clinical feature is severe, progressive lower limb spasticity. Nongenetic classification relies on characteristics such as mode of inheritance, age at onset, and the presence or absence of additional neurologic features. Several loci have been identified for autosomal dominant pure HSP. The most common form, which links to chromosome 2p (SPG4), has recently been shown to be due to mutations in spastin, the gene encoding a novel AAA‐containing protein. Results The authors report four generations of a British family with autosomal dominant HSP in whom haplotype analysis indicates linkage to chromosome 2p. In addition, a missense mutation has been identified in exon 10 of the spastin gene (A1395G). Dementia was documented clinically in one member of the family, two other affected family members were reported to have had late onset memory loss, and a younger affected individual showed evidence of memory disturbance and learning difficulties. Autopsy of the demented patient confirmed changes in the spinal cord typical of HSP and also demonstrated specific cortical pathology. There was neuronal depletion and tau‐immunoreactive neurofibrillary tangles in the hippocampus and tau‐immunoreactive balloon cells were seen in the limbic and neocortex. The substantia nigra showed Lewy body formation. The pathologic findings are not typical of known tauopathies. Conclusions The authors confirm that chromosome 2p‐linked HSP can be associated with dementia and that this phenotype may be associated with a specific and unusual cortical pathology.

Cloning of the mouse dysferlin gene and genomic characterization of the SJL-Dysf mutation.

The SJL mouse strain has been widely used as an animal model for experimental autoimmune encephalitis (EAE), inflammatory muscle disease and lymphomas and has also been used as a background strain for the generation of animal models for a variety of diseases including motor neurone disease, multiple sclerosis and atherosclerosis. Recently the SJL mouse was shown to have myopathy due to dysferlin deficiency, so that it can now be considered a natural animal model for limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM). We have cloned the mouse dysferlin cDNA and analysis of the sequence shows that the mouse dysferlin gene is characterized by six C2 domain sequences and a C-terminal anchoring domain, with the human and the mouse dysferlin genes sharing > 90% sequence homology overall. Genomic analysis of the SJL mutation confirms that the 171 bp RNA deletion has arisen by exon skipping resulting from a splice site mutation. The identification of this mutation has implications for the various groups using this widely available mouse stock.

A new distal myopathy with mutation in anoctamin 5

We have been following clinically and with muscle MRI for the past 3-decades a Finnish family with two patients with distal muscular dystrophy. Previously we demonstrated the cellular defect in these patients to be defective membrane repair and more recently have identified the causative gene to be anoctamin 5 (ANO5). The disorder seen in these patients is characterized by onset in the third decade. First symptoms were burning sensation on the calves and later on calf tightness during running. Muscle weakness and wasting were asymmetric and early involving the calf muscles, later spread to the thigh muscles. Biceps brachi was later manifestation. Clinical course was slow. CK levels were high. Muscle biopsy showed dystrophic pattern and multifocal disruption of the sarcolemmal membrane but no subsarcolemmal vesicle accumulation nor active inflammation. We conclude that the disease seen in our cases is a new separate clinical, genetic and histopathologic entity to include within the classification of autosomal recessive distal muscular dystrophies.

Lack of correlation between outcomes of membrane repair assay and correction of dystrophic changes in experimental therapeutic strategy in dysferlinopathy.

Mutations in the dysferlin gene are the cause of Limb-girdle Muscular Dystrophy type 2B and Miyoshi Myopathy. The dysferlin protein has been implicated in sarcolemmal resealing, leading to the idea that the pathophysiology of dysferlin deficiencies is due to a deficit in membrane repair. Here, we show using two different approaches that fullfiling membrane repair as asseyed by laser wounding assay is not sufficient for alleviating the dysferlin deficient pathology. First, we generated a transgenic mouse overexpressing myoferlin to test the hypothesis that myoferlin, which is homologous to dysferlin, can compensate for the absence of dysferlin. The myoferlin overexpressors show no skeletal muscle abnormalities, and crossing them with a dysferlin-deficient model rescues the membrane fusion defect present in dysferlin-deficient mice in vitro. However, myoferlin overexpression does not correct muscle histology in vivo. Second, we report that AAV-mediated transfer of a minidysferlin, previously shown to correct the membrane repair deficit in vitro, also fails to improve muscle histology. Furthermore, neither myoferlin nor the minidysferlin prevented myofiber degeneration following eccentric exercise. Our data suggest that the pathogenicity of dysferlin deficiency is not solely related to impairment in sarcolemmal repair and highlight the care needed in selecting assays to assess potential therapies for dysferlinopathies.