SMART MODE:

NORMAL GENOMIC

• Matsubara S, Kitaguchi T, Kawata A, Miyamoto K, Yagi H, Hirai S
• Experimental allergic myositis in SJL/J mouse. Reappraisal of immune reaction based on changes after single immunization.
• J Neuroimmunol. 2001; 119: 223-30
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• SJL/J mice have been subjected to immunization with wide varieties of antigens to produce models of autoimmune disorders including experimental myositis. They also have a defect in dysferlin gene and spontaneously develop muscle fiber degeneration, a condition akin to limb-girdle type muscular dystrophy and Miyoshi myopathy. To know whether muscle inflammation of SJL mice after immunization with muscle fractions really represents immune-mediated myositis or no more than an epiphenomenon of muscle degeneration due to dysferlin defect, we studied immunological parameters after immunization with rabbit myosin B fraction. Initial infiltration of macrophages and CD4+ lymphocytes on day 11 was followed by increase in number of CD8+ cells. Such increase was not observed in the nontreated and adjuvant controls. Some infiltrating cells were interferon gamma (IFN-gamma) positive. Furthermore, increased expression of the signal transducers and activator of transcription 1 (STAT-1) and interferon regulatory factor 1 (IRF-1) mRNA was shown in the first 2 weeks. These results indicate Th1 system activity in the muscle, rather than simple dysferlin deficiency, particularly 1-3 weeks after immunization. Thus it is concluded that an immune-mediated myositis is taking place at this stage. This model can be helpful in understanding pathomechanisms involved in the early stage of human myositides. It has also important implications concerning immune reactions associated with transplantation or gene therapy for muscular dystrophies.

• Mologni L, Salmikangas P, Fougerousse F, Beckmann JS, Carpen O
• Developmental expression of myotilin, a gene mutated in limb-girdle muscular dystrophy type 1A.
• Mech Dev. 2001; 103: 121-5
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• We analyzed developmental expression of myotilin, a novel sarcomeric component mutated in limb-girdle muscular dystrophy 1A (LGMD1A). In situ hybridization and immunostaining of embryonic mouse tissues revealed expression of myotilin initially (E9-10) in heart, somites and neuroepithelium. At E13 myotilin was expressed in a variety of tissues, including the nervous system, lung, liver and kidney, but upon organ differentiation expression became more restricted. The level of expression during early development is comparable between mouse and human, indicating that the mouse may provide a model for further studying the functions of myotilin and the pathogenesis of LGMD1A.

• Ueyama H et al.
• A new dysferlin gene mutation in two Japanese families with limb-girdle muscular dystrophy 2B and Miyoshi myopathy.
• Neuromuscul Disord. 2001; 11: 139-45
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• We found a new dysferlin gene mutation in two Japanese families, one with limb-girdle muscular dystrophy 2B and the other with Miyoshi myopathy. All patients in the limb-girdle muscular dystrophy 2B family showed apparent proximal dominant muscle atrophy and weakness, whereas a patient with Miyoshi myopathy in the second family showed distal muscle involvement at an early stage. The common clinical feature of all patients in both families was preferential involvement of calf muscles rather than the tibialis anterior muscle, which was confirmed by muscle computed tomography scan. All patients in both families shared the same homozygous alleles for chromosome 2p13 markers, and dysferlin gene analysis revealed a novel missense mutation, a G to A transition at nt 5882, which changed aspartic acid to asparagine at codon 1837. Allele-specific polymerase chain reaction analysis was used for confirmation of the mutation and for genotype analysis of the family members.

• Illa I et al.
• Distal anterior compartment myopathy: a dysferlin mutation causing a new muscular dystrophy phenotype.
• Ann Neurol. 2001; 49: 130-4
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• We report a family with a new phenotype of autosomal recessive muscle dystrophy caused by a dysferlin mutation. The onset of the illness is distal, in the muscles of the anterior compartment group. The disease is rapidly progressive, leading to severe proximal weakness. Muscle biopsy showed moderate dystrophic changes with no vacuoles. Dysferlin immunostaining was negative. Gene analysis revealed a frameshift mutation in the exon 50 (delG5966) of the DYSF gene. This phenotype further demonstrates the clinical heterogeneity of the dysferlinopathies.

• Aoki M et al.
• Genomic organization of the dysferlin gene and novel mutations in Miyoshi myopathy.
• Neurology. 2001; 57: 271-8
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• 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.

• Vafiadaki E et al.
• Cloning of the mouse dysferlin gene and genomic characterization of the SJL-Dysf mutation.
• Neuroreport. 2001; 12: 625-9
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• 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.

• Nakagawa M et al.
• Phenotypic variation in a large Japanese family with Miyoshi myopathy with nonsense mutation in exon 19 of dysferlin gene.
• J Neurol Sci. 2001; 184: 15-9
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• Miyoshi myopathy, an autosomal recessive muscular dystrophy involving distal muscles, is caused by dysferlin mutations. We present clinical and genetic studies of two men and six women, aged 25-83 years, from a Japanese family with consanguineous marriages. Onset was between ages 17 and 59 years. Six of the patients had muscle involvement typical of Miyoshi myopathy, one initially had severe proximal muscle involvement, and one had scapuloperoneal-type muscle involvement. Three patients showed steppage gait. Genetic linkage analysis identified a maximum lod score of 3.34 (θ=0.00) at marker D2S292 in 2p13. Analysis of dysferlin revealed the mutation G2090T (Glu573Stop) in exon 19 in all affected patients. This is the largest Japanese family with Miyoshi myopathy showing intrafamilial phenotypic variation and sharing a common mutation in dysferlin.

• Matsuda C et al.
• The sarcolemmal proteins dysferlin and caveolin-3 interact in skeletal muscle.
• Hum Mol Genet. 2001; 10: 1761-6
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• Dysferlin is a surface membrane protein in skeletal muscle whose deficiency causes distal and proximal, recessively inherited, forms of muscular dystrophy designated Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B), respectively. The function of dysferlin is not defined. Caveolin-3 is another skeletal muscle membrane protein which is important in the formation of caveolae and whose mutations cause dominantly inherited limb girdle muscular dystrophy type 1C (LGMD1C). We report that dysferlin co-immunoprecipitates with caveolin-3 from biopsied normal human skeletal muscles. We also describe abnormal localization of dysferlin in muscles from patients with LGMD1C including novel missense mutation (T64P) in the human caveolin-3 gene (CAV3). The immunoprecipitation data are consistent with the parallel observation that dysferlin immunostaining is not normal in LGMD1C muscles. Amino acid sequence analysis of the dysferlin protein reveals seven sites that correspond to caveolin-3 scaffold-binding motifs, and one site that is a potential target to bind the WW domain of the caveolin-3 protein. This is the first description of a possible dysferlin interacting protein; it suggests the hypothesis that one function of dysferlin may be to interact with caveolin-3 to subserve signaling functions of caveolae.

• Fanin M, Pegoraro E, Matsuda-Asada C, Brown RH Jr, Angelini C
• Calpain-3 and dysferlin protein screening in patients with limb-girdle dystrophy and myopathy.
• Neurology. 2001; 56: 660-5
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• BACKGROUND: Mutations in the genes encoding for calpain-3 and dysferlin are responsible for limb-girdle muscular dystrophy (LGMD) type 2A and 2B, the most common forms of autosomal recessive LGMD. OBJECTIVE: To identify calpain-3 or dysferlin deficiency in a large cohort of patients with as yet unclassified LGMD and myopathy through candidate protein analysis. METHODS: The authors' muscle biopsy database search identified 407 candidate muscle biopsies with normal dystrophin and sarcoglycan. Calpain-3 and dysferlin were studied by Western blotting and immunohistochemistry. RESULTS: Combined calpain-3 and dysferlin Western blot analysis identified calpain-3 deficiency in 66 (16%) muscle biopsies. In 31 cases (47%), the protein was absent, and in 35 (53%), it was severely reduced in amount (3 to 50% of control). Dysferlin deficiency was found in 26 (6.5%) muscle biopsies. In 9, the protein was absent (35%), and in 17 (65%), it was severely reduced in amount (traces to 20% of control). Twenty-eight percent (53/191) of patients with LGMD phenotype had calpain-3 deficiency. Sixty percent (21/35) of patients with distal myopathy had dysferlin deficiency. Dysferlin immunohistochemistry showed, in the completely dysferlin-deficient patients, absent reaction at the sarcolemma but positive nuclear membrane labeling and, in the partially dysferlin-deficient patients, scattered granular positive cytoplasmic areas and diffuse reaction in regenerating fibers. CONCLUSION: About 25% of previously unclassified dystrophy/myopathy cases are due to calpain-3 or dysferlin protein deficiency. These results suggest that immunoblot analysis may be used to define patients for calpain-3 and dysferlin gene mutation studies.

• Betz RC et al.
• Mutations in CAV3 cause mechanical hyperirritability of skeletal muscle in rippling muscle disease.
• Nat Genet. 2001; 28: 218-9
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• Hereditary rippling muscle disease (RMD) is an autosomal dominant human disorder characterized by mechanically triggered contractions of skeletal muscle. Genome-wide linkage analysis has identified an RMD locus on chromosome 3p25. We found missense mutations in positional candidate CAV3 (encoding caveolin 3; ref. 5) in all five families analyzed. Mutations in CAV3 have also been described in limb-girdle muscular dystrophy type 1C (LGMD1C; refs. 6,7), demonstrating the allelism of dystrophic and non-dystrophic muscle diseases.

• Illarioshkin SN et al.
• Identical dysferlin mutation in limb-girdle muscular dystrophy type 2B and distal myopathy.
• Neurology. 2000; 55: 1931-3
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• Limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM) are autosomal recessive disorders caused by mutations in the dysferlin gene on chromosome 2p13. The authors studied a large Russian family with both LGMD2B and MM. All affected individuals, as well as one preclinical boy with dystrophic changes on muscle biopsy, were found to be homozygous for a novel dysferlin mutation, TG573/574AT (Val67Asp). This finding supports the view that additional factors (e.g., modifier genes) contribute to the phenotypic expression of causative mutations in dysferlinopathies.

• Anderson LV et al.
• Secondary reduction in calpain 3 expression in patients with limb girdle muscular dystrophy type 2B and Miyoshi myopathy (primary dysferlinopathies).
• Neuromuscul Disord. 2000; 10: 553-9
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• 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.

• Rich T, Allen RL, Lucas AM, Stewart A, Trowsdale J
• Pellino-related sequences from Caenorhabditis elegans and Homo sapiens.
• Immunogenetics. 2000; 52: 145-9

• Sorimachi H, Ono Y, Suzuki K
• Molecular analysis of p94 and its application to diagnosis of limb girdle muscular dystrophy type 2A.
• Methods Mol Biol. 2000; 144: 75-84

• Driss A et al.
• A new locus for autosomal recessive limb-girdle muscular dystrophy in a large consanguineous Tunisian family maps to chromosome 19q13.3.
• Neuromuscul Disord. 2000; 10: 240-6
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• Autosomal recessive limb-girdle muscular dystrophies represent a genetically heterogeneous group of diseases characterized by a progressive involvement of skeletal muscles. They show a wide spectrum of clinical courses, varying from very mild to severe. Eight loci responsible for autosomal recessive limb-girdle muscular dystrophies have been mapped and six defective genes identified. In this study, we report the clinical data, muscle biopsy findings and results of genetic linkage analysis in a large consanguineous Tunisian family with 13 individuals suffering from autosomal recessive limb-girdle muscular dystrophy. Clinical features include variable age of onset, proximal limb muscle weakness and wasting predominantly affecting the pelvic girdle, and variable course between siblings. CK rate was usually high in younger patients. Muscle biopsy showed dystrophic changes with normal expression of dystrophin and various proteins of the dystrophin-associated protein complex (sarcoglycan sub-units, dystroglycan, and sarcospan). Genetic linkage analysis excluded the known limb-girdle muscular dystrophies loci as well as ten additional candidate genes. A maximum LOD score of 4.36 at θ=0.00 was obtained with marker D19S606, mapping this new form of autosomal recessive limb-girdle muscular dystrophy to chromosome 19q13.3.

• Illa I
• Distal myopathies.
• J Neurol. 2000; 247: 169-74
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• Distal myopathies are classified according to clinical, histopathological, and genetic patterns into the following: late adult onset type 1, or Welander myopathy, the first recognized distal myopathy with autosomal dominant inheritance and very recently linked to chromosome 2p; late adult onset type 2, or Markesbery-Griggs/Udd myopathy, autosomal dominant with linkage to chromosome 2q; early adult onset type 1, or Nonaka myopathy, an autosomal recessive disease linked to 9p1-q1 and considered indistinguishable from hereditary inclusion body myopathy; early adult onset type 2, or Miyoshi myopathy, with autosomal recessive inheritance linked to chromosome 2p12-p14; and early adult onset type 3, or Laing myopathy, autosomal dominant with linkage to chromosome 14. Very recently, dysferlin, a novel skeletal muscle gene, has been found mutated in Miyoshi myopathy and also in the limb girdle muscular dystrophy 2B, a disease with a completely different phenotype. This indicates that the classification of the distal and other genetically determined muscle diseases will probably change when these myopathies are understood at the molecular level. For example, it would be reasonable to use the term dysferlinopathies to describe all the diseases due to dysferlin mutations.

• Herrmann R et al.
• Dissociation of the dystroglycan complex in caveolin-3-deficient limb girdle muscular dystrophy.
• Hum Mol Genet. 2000; 9: 2335-40
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• 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.

• Zatz M, Vainzof M, Passos-Bueno MR
• Limb-girdle muscular dystrophy: one gene with different phenotypes, one phenotype with different genes.
• Curr Opin Neurol. 2000; 13: 511-7
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• Among 14 limb-girdle muscular dystrophy genes that have been mapped, 10 (three autosomal dominant and seven autosomal recessive) have so far had their product identified. This review will focus on the most recent data in the field and on our own experience of more than 200 patients studied with autosomal recessive-limb-girdle muscular dystrophy, classified from calpainopathy to telethoninopathy. Genotype: phenotype correlations in this highly heterogeneous group show a similar clinical course among patients with different forms, whereas a discordant phenotype may be seen in unrelated patients or in affected sibs carrying the same mutation. Understanding such similarities or differences remains a major challenge. It will depend on future knowledge of gene-protein functions, on protein interactions and on identifying modifying genes and other factors underlying clinical variability.

• Moreira ES et al.
• Limb-girdle muscular dystrophy type 2G is caused by mutations in the gene encoding the sarcomeric protein telethonin.
• Nat Genet. 2000; 24: 163-6
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• Autosomal recessive limb-girdle muscular dystrophies (AR LGMDs) are a genetically heterogeneous group of disorders that affect mainly the proximal musculature. There are eight genetically distinct forms of AR LGMD, LGMD 2A-H (refs 2-10), and the genetic lesions underlying these forms, except for LGMD 2G and 2H, have been identified. LGMD 2A and LGMD 2B are caused by mutations in the genes encoding calpain 3 (ref. 11) and dysferlin, respectively, and are usually associated with a mild phenotype. Mutations in the genes encoding gamma-(ref. 14), alpha-(ref. 5), beta-(refs 6,7) and delta (ref. 15)-sarcoglycans are responsible for LGMD 2C to 2F, respectively. Sarcoglycans, together with sarcospan, dystroglycans, syntrophins and dystrobrevin, constitute the dystrophin-glycoprotein complex (DGC). Patients with LGMD 2C-F predominantly have a severe clinical course. The LGMD 2G locus maps to a 3-cM interval in 17q11-12 in two Brazilian families with a relatively mild form of AR LGMD (ref. 9). To positionally clone the LGMD 2G gene, we constructed a physical map of the 17q11-12 region and refined its localization to an interval of 1.2 Mb. The gene encoding telethonin, a sarcomeric protein, lies within this candidate region. We have found that mutations in the telethonin gene cause LGMD 2G, identifying a new molecular mechanism for AR LGMD.

• Barresi R, Moore SA, Stolle CA, Mendell JR, Campbell KP
• Expression of gamma -sarcoglycan in smooth muscle and its interaction with the smooth muscle sarcoglycan-sarcospan complex.
• J Biol Chem. 2000; 275: 38554-60
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• The sarcoglycan complex in striated muscle is a heterotetrameric unit integrally associated with sarcospan in the dystrophin-glycoprotein complex. The sarcoglycans, alpha, beta, gamma, and delta, are mutually dependent with regard to their localization at the sarcolemma, and mutations in any of the sarcoglycan genes lead to limb-girdle muscular dystrophies type 2C-2F. In smooth muscle beta- and delta-sarcoglycans are associated with epsilon-sarcoglycan, a glycoprotein homologous to alpha-sarcoglycan. Here, we demonstrate that gamma-sarcoglycan is also a component of the sarcoglycan complex in the smooth muscle. First, we show the presence of gamma-sarcoglycan in a number of smooth muscle-containing organs, and we verify the existence of identical transcripts in skeletal and smooth muscle. The specificity of the expression of gamma-sarcoglycan in smooth muscle was confirmed by analysis of smooth muscle cells in culture. Next, we provide evidence for the association of gamma-sarcoglycan with the sarcoglycan-sarcospan complex by biochemical analysis and comparison among animal models for muscular dystrophy. Moreover, we find disruption of the sarcoglycan complex in the vascular smooth muscle of a patient with gamma-sarcoglycanopathy. Taken together, our results prove that the sarcoglycan complex in vascular and visceral smooth muscle consists of epsilon-, beta-, gamma-, and delta-sarcoglycans and is associated with sarcospan.

• Piccolo F, Moore SA, Ford GC, Campbell KP
• Intracellular accumulation and reduced sarcolemmal expression of dysferlin in limb--girdle muscular dystrophies.
• Ann Neurol. 2000; 48: 902-12
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• Dysferlin has recently been identified as a novel gene involved in limb-girdle muscular dystrophy type 2B (LGMD2B) and its allelic disease, Miyoshi myopathy. The predicted structure of dysferlin suggests that it is a transmembrane protein possibly involved in membrane fusion. Thus, unlike previously identified structural proteins in muscular dystrophy, dysferlin is likely involved in a novel pathogenic mechanism for this disease. In this study, we have analyzed the expression of dysferlin in skeletal muscle of patients with disruptions in the dystrophin-glycoprotein complex and patients with a clinical diagnosis of LGMD2B or Miyoshi myopathy. We show expression of dysferlin at the sarcolemma in normal muscle and reduced sarcolemmal expression along with accumulation of intracellular staining in dystrophic muscle. Electron microscopy in Miyoshi myopathy biopsies suggests that the cytoplasmic staining could be a result of the abundance of intracellular vesicles. Our results indicate that dysferlin expression is perturbed in LGMD and that both mutations in the dysferlin gene and disruption of the dystrophin-glycoprotein complex can lead to the accumulation of dysferlin within the cytoplasm.

• Argov Z et al.
• Muscular dystrophy due to dysferlin deficiency in Libyan Jews. Clinical and genetic features.
• Brain. 2000; 123: 1229-37
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• The cluster in Jews of Libyan origin of limb-girdle muscular dystrophy type 2B due to a dysferlin 1624delG mutation is described. The carrier frequency of this mutation is calculated to be approximately 10% in this population, in which the disease prevalence is at least 1 per 1300 adults. Twenty-nine patients from 12 families were all homozygous for the same mutation. However, clinical features were heterogeneous even within the same family: in half of the patients onset was in the distal muscles of the legs, which is similar to Miyoshi myopathy, while in others onset was in the proximal musculature, which is similar to other forms of limb-girdle dystrophies. Age at onset varied from 12 to 28 years (mean 20.3 +/- 5.5 years). One patient was presymptomatic at age 28 years. Progression was slow regardless of age of onset, patients remaining ambulatory until at least 33 years. Five patients described subacute, painful enlarged calves as an early, unusual feature. The variable features in this ethnic cluster contribute to the definition of the clinical spectrum of dysferlinopathies in general. The cause of the observed heterogeneity remains unclear.

• Sorimachi H, Ono Y, Suzuki K
• Skeletal muscle-specific calpain, p94, and connectin/titin: their physiological functions and relationship to limb-girdle muscular dystrophy type 2A.
• Adv Exp Med Biol. 2000; 481: 383-95
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• The skeletal muscle-specific calpain homologue, p94 (also called calpain 3), is essential for normal muscle function. A mutation of the p94 gene causes limb-girdle muscular dystrophy type 2A (LGMD2A), which is one type of autosomal recessive inherited disease characterized by progressive muscular degeneration. In myofibrils, p94 specifically binds to connectin/titin, and the activity of p94 is probably suppressed by this binding. Thus, we postulate that a signal transduction pathway exists, involving p94 and connectin/titin to modulate functions of skeletal muscle, and LGMD2A occurs when this signalling pathway is not properly regulated by p94. LGMD2A mutants of p94 also reveal significant information on the factors that relate structure to function in this molecule.

• Culetto E, Sattelle DB
• A role for Caenorhabditis elegans in understanding the function and interactions of human disease genes.
• Hum Mol Genet. 2000; 9: 869-77
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• A growing number of medical research teams have begun to explore the experimental advantages of using a genetic animal model, the nematode worm Caenorhabditis elegans, with a view to enhancing our understanding of genes underlying human congenital disorders. In this study, we have compared sequences of positionally cloned human disease genes with the C.elegans database of predicted genes. Drawing on examples from spinal muscular atrophy, polycystic kidney disease, muscular dystrophy and Alzheimer's disease, we illustrate how data from C.elegans can yield new insights into the function and interactions of human disease genes.

• Plenefisch JD, Zhu X, Hedgecock EM
• Fragile skeletal muscle attachments in dystrophic mutants of Caenorhabditis elegans: isolation and characterization of the mua genes.
• Development. 2000; 127: 1197-207
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• Over 30 Caenorhabditis elegans mutants were identified with normal muscle differentiation and initial locomotion followed by catastrophic detachment of skeletal muscles from the body wall. Reducing the strength of muscle contraction in these mutants with a myosin gene mutation suppresses muscle detachment. These dystrophic mutants identify a novel class of genes required for growth and maintenance of functional muscle attachments, not exceptional alleles of genes required for muscle differentiation and contractility. Nine new genes, named mua, and two previously published loci, unc-23 and vab-10, cause fragile musscle attachments. The primary sites of muscle detachment, including the plane of tissue separation, are characteristic for each gene. We suggest these genes identify feedback mechanisms whereby local strain regulates the extent of myofibril contraction and the placement of new muscle attachments in functioning muscles. Finally, we draw some comparisons to vertebrate skin fragility diseases and muscular dystrophies.

• Carbone I et al.
• Mutation in the CAV3 gene causes partial caveolin-3 deficiency and hyperCKemia.
• Neurology. 2000; 54: 1373-6
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• Mutations in the caveolin-3 (CAV3) gene are associated with autosomal dominant limb-girdle muscular dystrophy (LGMD1C). The authors report a novel sporadic mutation in the CAV3 gene in two unrelated children with persistent elevated levels of serum creatine kinase (hyperCKemia) without muscle weakness. Immunohistochemistry and quantitative immunoblot analysis of caveolin-3 showed reduced expression of the protein in muscle fibers. Our data indicate that a partial caveolin-3 deficiency should be considered in the differential diagnosis of idiopathic hyperCKemia.

• Bornemann A, Anderson LV
• Diagnostic protein expression in human muscle biopsies.
• Brain Pathol. 2000; 10: 193-214
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• Using immunohistochemistry in diagnosing neuromuscular diseases is meant to enhance the diagnostic yield in two ways. The first application aims at visualizing molecules which are developmentally, neurally, and/or immunologically regulated and not expressed by normal muscle. They are upregulated in pathological conditions and may help assign a given muscular biopsy to one of the main diagnostic entities (muscular dystrophies, inflammatory myopathy, neurogenic atrophy). In the past, muscle-specific molecules with a defined expression pattern during fetal myogenesis served as antigens, with the rationale that the developmental program was switched on in new fibers. Recently, myofibers in diseased muscle are thought of as targets of stimuli which are released by macrophages in muscular dystrophy, by lymphocytes in inflammatory myopathies, or by a lesioned peripheral nerve in neurogenic atrophies. This has somewhat blurred the borders between the diagnostic groups, for certain molecules, e.g. cytokines, may be upregulated after experimental necrotization, denervation, and also in inflammatory myopathies. In the second part of this review we summarise the experiences of a Centre in the North of England that specialises in the diagnosis and clinical support of patients with muscular dystrophy. Emphasis is placed on the use of protein expression to guide mutation analysis, particularly in the limb-girdle muscular dystrophies (a group of diseases that are very difficult to differentiate on clinical grounds alone). We confirm that genetic analysis is essential to corroborate the results of protein analysis in certain conditions (particularly in calpainopathy). However, we conclude that analysing biopsies for abnormal protein expression is very useful in aiding the decision between alternative diagnoses.

• Muchir A et al.
• Identification of mutations in the gene encoding lamins A/C in autosomal dominant limb girdle muscular dystrophy with atrioventricular conduction disturbances (LGMD1B).
• Hum Mol Genet. 2000; 9: 1453-9
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• LGMD1B is an autosomal dominantly inherited, slowly progressive limb girdle muscular dystrophy, with age-related atrioventricular cardiac conduction disturbances and the absence of early contractures. The disease has been linked to chromosome 1q11-q21. Within this locus another muscular dystrophy, the autosomal dominant form of Emery-Dreifuss muscular dystrophy (AD-EDMD) has recently been mapped and the corresponding gene identified. AD-ADMD is characterized by early contractures of elbows and Achilles tendons and a humero-peroneal distribution of weakness combined with a cardiomyopathy with conduction defects. The disease gene of AD-EDMD is LMNA which encodes lamins A/C, two proteins of the nuclear envelope. In order to identify whether or not LGMD1B and AD-EDMD are allelic disorders, we carried out a search for mutations in the LMNA gene in patients with LGMD1B. For this, PCR/SSCP/sequencing screening was carried out for the 12 exons of LMNA on DNA samples of individuals from three LGMD1B families that were linked to chromo-some 1q11-q21. Mutations were identified in all three LGMD1B families: a missense mutation, a deletion of a codon and a splice donor site mutation, respectively. The three mutations were identified in all affected members of the corresponding families and were absent in 100 unrelated control subjects. The present identification of mutations in the LMNA gene in LGMD1B demonstrates that LGMD1B and AD-EDMD are allelic disorders. Further analysis of phenotype-genotype relationship will help to clarify the variability of the phenotype observed in these two muscular dystrophies.

• McNally EM et al.
• Splicing mutation in dysferlin produces limb-girdle muscular dystrophy with inflammation.
• Am J Med Genet. 2000; 91: 305-12
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• Mutations in dysferlin were recently described in patients with Miyoshi myopathy, a disorder that preferentially affects the distal musculature, and in patients with Limb-Girdle Muscular Dystrophy 2B, a disorder that affects the proximal musculature. Despite the phenotypic differences, the types of mutations associated with Miyoshi myopathy and Limb-Girdle Muscular Dystrophy 2B do not differ significantly. Thus, the etiology of the phenotypic variability associated with dysferlin mutations remains unknown. Using genetic linkage and mutation analysis, we identified a large inbred pedigree of Yemenite Jewish descent with limb-girdle muscular dystrophy. The phenotype in these patients included slowly progressive, proximal, and distal muscular weakness in the lower limbs with markedly elevated serum creatine kinase (CK) levels. These patients had normal development and muscle strength and function in early life. Muscle biopsies from 4 affected patients showed a typical dystrophic pattern but interestingly, in 2, an inflammatory process was seen. The inflammatory infiltrates included primarily CD3 positive lymphocytes. Associated with this phenotype, we identified a previously undescribed frameshift mutation at nucleotide 5711 of dysferlin. This mutation produced an absence of normal dysferlin mRNA synthesis by affecting an acceptor site and cryptic splicing. Thus, splice site mutations that disrupt dysferlin may produce a phenotype associated with inflammation.

• Davis DB, Delmonte AJ, Ly CT, McNally EM
• Myoferlin, a candidate gene and potential modifier of muscular dystrophy.
• Hum Mol Genet. 2000; 9: 217-26
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• Dysferlin, the gene product of the limb girdle muscular dystrophy (LGMD) 2B locus, encodes a membrane-associated protein with homology to Caenorhabditis elegans fer-1. Humans with mutations in dysferlin ( DYSF ) develop muscle weakness that affects both proximal and distal muscles. Strikingly, the phenotype in LGMD 2B patients is highly variable, but the type of mutation in DYSF cannot explain this phenotypic variability. Through electronic database searching, we identified a protein highly homologous to dysferlin that we have named myoferlin. Myoferlin mRNA was highly expressed in cardiac muscle and to a lesser degree in skeletal muscle. However, antibodies raised to myoferlin showed abundant expression of myoferlin in both cardiac and skeletal muscle. Within the cell, myoferlin was associated with the plasma membrane but, unlike dysferlin, myoferlin was also associated with the nuclear membrane. Ferlin family members contain C2 domains, and these domains play a role in calcium-mediated membrane fusion events. To investigate this, we studied the expression of myoferlin in the mdx mouse, which lacks dystrophin and whose muscles undergo repeated rounds of degeneration and regeneration. We found upregulation of myoferlin at the membrane in mdx skeletal muscle. Thus, myoferlin ( MYOF ) is a candidate gene for muscular dystrophy and cardiomyopathy, or possibly a modifier of the muscular dystrophy phenotype.

• Hauser MA et al.
• Myotilin is mutated in limb girdle muscular dystrophy 1A.
• Hum Mol Genet. 2000; 9: 2141-7
• Display abstract

• We have identified a mutation in the myotilin gene in a large North American family of German descent expressing an autosomal dominant form of limb girdle muscular dystrophy (LGMD1A). We have previously mapped this gene to 5q31. Symptoms of this adult onset disease are progressive weakness of the hip and shoulder girdles, as well as a distinctive dysarthric pattern of speech. Muscle of affected individuals shows degeneration of myofibers, variations in fiber size, fiber splitting, centrally located myonuclei and a large number of autophagic vesicles. Affected muscle also exhibits disorganization and streaming of the Z-line similar to that seen in nemaline myopathy. We have identified a C450T missense mutation in the myotilin gene that is predicted to result in the conversion of residue 57 from threonine to isoleucine. This mutation has not been found in 396 control chromosomes. The mutant allele is transcribed and normal levels of correctly localized myotilin protein are seen in LGMD1A muscle. Myotilin is a sarcomeric protein that binds to alpha-actinin and is localized in the Z-line. The observed missense mutation does not disrupt binding to alpha-actinin.

• Razani B, Schlegel A, Lisanti MP
• Caveolin proteins in signaling, oncogenic transformation and muscular dystrophy.
• J Cell Sci. 2000; 113: 2103-9
• Display abstract

• In adult animals and humans, signal transduction maintains homeostasis. When homeostatic mechanisms are interrupted, an illness or disease may ensue. Caveolae are plasma membrane specializations that contain the structural proteins caveolins, and appear to be important for normal signal transduction. The caveolin scaffolding domain interacts with several signaling molecules, sequestering them in the absence of activating signals, and thereby reducing the signal-to-noise ratio. Deletion and mutation of genes that encode caveolins is implicated in the pathogenesis of several human diseases. Down-regulation of caveolin-1 protein expression leads to deregulated signaling and consequently tumorigenesis, whereas naturally occurring dominant-negative caveolin-3 mutations cause muscular dystrophy.

• Speer MC et al.
• Identification of a new autosomal dominant limb-girdle muscular dystrophy locus on chromosome 7.
• Am J Hum Genet. 1999; 64: 556-62
• Display abstract

• We report the identification of a new locus for autosomal dominant limb-girdle muscular dystrophy (LGMD1) on 7q. Two of five families (1047 and 1701) demonstrate evidence in favor of linkage to this region. The maximum two-point LOD score for family 1047 was 3.76 for D7S427, and that for family 1701 was 2.63 for D7S3058. Flanking markers place the LGMD1 locus between D7S2423 and D7S427, with multipoint analysis slightly favoring the 9-cM interval spanned by D7S2546 and D7S2423. Three of five families appear to be unlinked to this new locus on chromosome 7, thus establishing further heterogeneity within the LGMD1 diagnostic classification.

• Yasunaga S et al.
• A mutation in OTOF, encoding otoferlin, a FER-1-like protein, causes DFNB9, a nonsyndromic form of deafness.
• Nat Genet. 1999; 21: 363-9
• Display abstract

• Using a candidate gene approach, we identified a novel human gene, OTOF, underlying an autosomal recessive, nonsyndromic prelingual deafness, DFNB9. The same nonsense mutation was detected in four unrelated affected families of Lebanese origin. OTOF is the second member of a mammalian gene family related to Caenorhabditis elegans fer-1. It encodes a predicted cytosolic protein (of 1,230 aa) with three C2 domains and a single carboxy-terminal transmembrane domain. The sequence homologies and predicted structure of otoferlin, the protein encoded by OTOF, suggest its involvement in vesicle membrane fusion. In the inner ear, the expression of the orthologous mouse gene, mainly in the sensory hair cells, indicates that such a role could apply to synaptic vesicles.

• Bushby KM
• The limb-girdle muscular dystrophies-multiple genes, multiple mechanisms.
• Hum Mol Genet. 1999; 8: 1875-82
• Display abstract

• In the field of muscular dystrophy, advances in understanding the molecular basis of the various disorders in this group have been rapidly translated into readily applicable diagnostic tests, allowing the provision of more accurate prognostic and genetic counselling. The limb-girdle muscular dystrophies (LGMD) have recently undergone a major reclassification according to their genetic basis. Currently 13 different types can be recognized. Amongst this group, increasing diversity of the mechanisms involved in producing a muscular dystrophy phenotype is emerging. Recent insights into the involvement of the dystrophin glycoprotein complex in muscular dystrophy suggests that its members may play distinct or even multiple roles in the maintenance of muscle fibre integrity. In other forms of LGMD, proteins have been implicated which may be important in intracellular signalling, vesicle trafficking or the control of transcription. As these various mechanisms are more fully elucidated, further insights will be gained into the pathophysiology of muscular dystrophy. At a practical level, despite the marked heterogeneity of this group real progress can at last be made in determining a precise diagnosis.

• Toda T
• [Fukutin, a novel protein product responsible for Fukuyama-type congenital muscular dystrophy]
• Seikagaku. 1999; 71: 55-61

• Bittner RE et al.
• Dysferlin deletion in SJL mice (SJL-Dysf) defines a natural model for limb girdle muscular dystrophy 2B.
• Nat Genet. 1999; 23: 141-2

• Anderson LV et al.
• Dysferlin is a plasma membrane protein and is expressed early in human development.
• Hum Mol Genet. 1999; 8: 855-61
• Display abstract

• Recently, a single gene, DYSF, has been identified which is mutated in patients with limb-girdle muscular dystrophy type 2B (LGMD2B) and with Miyoshi myopathy (MM). This is of interest because these diseases have been considered as two distinct clinical conditions since different muscle groups are the initial targets. Dysferlin, the protein product of the gene, is a novel molecule without homology to any known mammalian protein. We have now raised a monoclonal antibody to dysferlin and report on the expression of this new protein: immunolabelling with the antibody (designated NCL-hamlet) demonstrated a polypeptide of approximately 230 kDa on western blots of skeletal muscle, with localization to the muscle fibre membrane by microscopy at both the light and electron microscopic level. A specific loss of dysferlin labelling was observed in patients with mutations in the LGMD2B/MM gene. Furthermore, patients with two different frameshifting mutations demonstrated very low levels of immunoreactive protein in a manner reminiscent of the dystrophin expressed in many Duchenne patients. Analysis of human fetal tissue showed that dysferlin was expressed at the earliest stages of development examined, at Carnegie stage 15 or 16 (embryonic age 5-6 weeks). Dysferlin is present, therefore, at a time when the limbs start to show regional differentiation. Lack of dysferlin at this critical time may contribute to the pattern of muscle involvement that develops later, with the onset of a muscular dystrophy primarily affecting proximal or distal muscles.

• Betto R, Biral D, Sandona D
• Functional roles of dystrophin and of associated proteins. New insights for the sarcoglycans.
• Ital J Neurol Sci. 1999; 20: 371-9
• Display abstract

• The discovery of the dystrophin gene, whose mutations lead to Duchenne's and Becker's muscular dystrophy (DMD and BMD), represents the first important landmark by which, in the last ten years, molecular biology and genetic studies have revealed many of the molecular defects of the major muscular dystrophies. Very rapidly, several studies revealed the presence at skeletal and cardiac muscle sarcolemma of a group of proteins associated to dystrophin. This includes a set of five transmembrane glycoproteins, the sarcoglycans, whose physiological role, however, is still poorly understood. Dystrophin and the associated proteins are believed to play an important role in membrane stability and maintenance during muscle contraction and relaxation. However, the absence of sarcoglycans from sarcolemma does not appear to affect membrane integrity suggesting that these components of the dystrophin complex are recipients of other important functions. This review deals with recent advances in the knowledge of sarcoglycan function and organization that may give important insights into the pathogenetic mechanisms of muscular dystrophies.

• Weiler T et al.
• Identical mutation in patients with limb girdle muscular dystrophy type 2B or Miyoshi myopathy suggests a role for modifier gene(s).
• Hum Mol Genet. 1999; 8: 871-7
• Display abstract

• Limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM), a distal muscular dystrophy, are both caused by mutations in the recently cloned gene dysferlin, gene symbol DYSF. Two large pedigrees have been described which have both types of patient in the same families. Moreover, in both pedigrees LGMD2B and MM patients are homozygous for haplotypes of the critical region. This suggested that the same mutation in the same gene would lead to both LGMD2B or MM in these families and that additional factors were needed to explain the development of the different clinical phenotypes. In the present paper we show that in one of these families Pro791 of dysferlin is changed to an Arg residue. Both the LGMD2B and MM patients in this kindred are homozygous for this mutation, as are four additional patients from two previously unpublished families. Haplotype analyses suggest a common origin of the mutation in all the patients. On western blots of muscle, LGMD2B and MM patients show a similar abundance in dysferlin staining of 15 and 11%, respectively. Normal tissue sections show that dysferlin localizes to the sarcolemma while tissue sections from MM and LGMD patients show minimal staining which is indistinguishable between the two types. These findings emphasize the role for the dysferlin gene as being responsible for both LGMD2B and MM, but that the distinction between these two clinical phenotypes requires the identification of additional factor(s), such as modifier gene(s).

• Salmikangas P, Mykkanen OM, Gronholm M, Heiska L, Kere J, Carpen O
• Myotilin, a novel sarcomeric protein with two Ig-like domains, is encoded by a candidate gene for limb-girdle muscular dystrophy.
• Hum Mol Genet. 1999; 8: 1329-36
• Display abstract

• The striated muscle sarcomeres are highly organized structures composed of actin (thin) and myosin (thick) filaments that slide past each other during contraction. The integrity of sarcomeres is controlled by a set of structural proteins, among which are titin, a giant molecule that contains several immunoglobulin (Ig)-like domains and associates with thin and thick filaments, and [alpha]-actinin, an actin cross-linking protein. Mutations in several sarcomeric and sarcolemmal proteins have been shown to result in muscular dystrophy and cardiomyopathy. On the other hand, the disease genes underlying several disease forms remain to be identified. Here we describe a novel 57 kDa cytoskeletal protein, myotilin. Its N-terminal sequence is unique, but the C-terminal half contains two Ig-like domains homologous to titin. Myotilin is expressed in skeletal and cardiac muscle, it co-localizes with [alpha]-actinin in the sarcomeric I--bands and directly interacts with [alpha]-actinin. The human myotilin gene maps to chromosome 5q31 between markers AFM350yB1 and D5S500. The locus of a dominantly inherited limb-girdle muscular dystrophy (LGMD1A) resides in an overlapping narrow segment, and a new type of distal myopathy with vocal cord and pharyngeal weakness (VCPMD) has been mapped to the same locus. The muscle specificity and apparent role as a sarcomeric structural protein raise the possibility that defects in the myotilin gene may cause muscular dystrophy.

• Gieseler K, Bessou C, Segalat L
• Dystrobrevin- and dystrophin-like mutants display similar phenotypes in the nematode Caenorhabditis elegans.
• Neurogenetics. 1999; 2: 87-90
• Display abstract

• Dystrophin, the protein disrupted in Duchenne muscular dystrophy, forms a transmembrane complex with dystrophin-associated proteins. Dystrobrevins, proteins showing homology to the C-terminal end of dystrophin, and whose function is unknown, are part of the dystrophin complex. We report here that, in the nematode Caenorhabditis elegans, animals carrying mutations in either the dystrophin-like gene dys-1 or the dystrobrevin-like gene dyb-1 display similar behavioral and pharmacological phenotypes consistent with an alteration of cholinergic signalling. These findings suggest that: (1) dystrobrevin and dystrophin are functionally related and (2) their disruption impairs cholinergic signalling.

• Nevo Y, Yaron Y
• [Update on diagnosis of limb girdle muscular dystrophy]
• Harefuah. 1999; 136: 720-5

• Gieseler K, Abdel-Dayem M, Segalat L
• In vitro interactions of Caenorhabditis elegans dystrophin with dystrobrevin and syntrophin.
• FEBS Lett. 1999; 461: 59-62
• Display abstract

• Dystrophin, the product of the gene mutated in Duchenne muscular dystrophy (DMD) is bound by its C-terminus to a protein complex including the related protein dystrobrevin. Both proteins contain a putative coiled-coil domain consisting of two alpha-helices. It has been reported that the two proteins bind to each other by the first one of the two alpha-helices. We have revisited this question using the Caenorhabditis elegans homologs of dystrophin and dystrobrevin. In vitro interaction occurs through the more conserved second helix. We propose a new model of dystrophin interactions with associated proteins.

• Aoki M, Arahata K, Brown RH Jr
• [Positional cloning of the gene for Miyoshi myopathy and limb-girdle muscular dystrophy]
• Rinsho Shinkeigaku. 1999; 39: 1272-5
• Display abstract

• Miyoshi myopathy (MM) is autosomal recessive distal muscular dystrophy that we have mapped to chromosome 2 p13. We constructed a 3 Mb P 1-derived artificial chromosome contig spanning the MM candidate region. Using this and new polymorphic markers within it, we recently identified a novel, full-length 6.9 kb muscle cDNA, whose corresponding protein we designated "dysferlin" (Nature Genet, 1998: 20: 31-36). We described eighteen mutations in the dysferlin gene with MM or limb-girdle muscular dystrophy type 2 B (LGMD 2 B). Most are predicted to block translation of dysferlin protein. In some cases, corresponding Western immunoblotting reveals absence of dysferlin in muscle biopsies. Identical mutations in the dysferlin gene can produce more than one myopathy phenotype (MM, limb-girdle dystrophy, distal myopathy with anterior tibial onset).

• Matsuda C, Aoki M, Hayashi YK, Ho MF, Arahata K, Brown RH Jr
• Dysferlin is a surface membrane-associated protein that is absent in Miyoshi myopathy.
• Neurology. 1999; 53: 1119-22
• Display abstract

• Recently we reported that mutations in a muscle protein "dysferlin" are present in limb girdle muscular dystrophy-2B and a related, adult-onset, distal dystrophy known as Miyoshi myopathy (MM). We report that antibodies to dysferlin identify a protein of approximately 230 kDa and show that dysferlin is located in the muscle membrane. This protein is absent in MM and LGMD-2B muscle. By contrast, dystrophin and other dystrophin-associated proteins are normal in these patients. Thus, dysferlin is a membrane-associated protein that is not likely to be an integral component of the dystrophin complex. Although it is not essential for initial myogenesis, it appears to be critical for sustained normal function in mature muscle.

• Bushby KM
• Making sense of the limb-girdle muscular dystrophies.
• Brain. 1999; 122: 1403-20
• Display abstract

• The clinical heterogeneity which has long been recognized in the limb-girdle muscular dystrophies (LGMD) has been shown to relate to the involvement of a large number of different genes. At least eight forms of autosomal recessive LGMD and three forms of autosomal dominant disease are now recognized and can be defined by the primary gene or protein involved, or by a genetic localization. These advances have combined the approaches of positional cloning and candidate gene analysis to great effect, with the pivotal role of the dystrophin-associated complex confirmed through the involvement of at least four dystrophin-associated proteins in different subtypes of autosomal recessive LGMD (the sarcoglycanopathies). Two novel mechanisms may have to be postulated to explain the involvement of the calpain 3 and dysferlin genes in other forms of LGMD. Using the diagnostic tools which have become available as a result of this increased understanding, the clinical features of the various subtypes are also becoming clearer, with useful diagnostic and prognostic information at last available to the practising clinician.

• Anderson LV
• Immunomarkers for molecular mass.
• Neuromuscul Disord. 1999; 9: 421-2
• Display abstract

• The proteins known to cause different forms of muscular dystrophy cover a wide range of sizes, and it is now possible to use a cocktail of antibodies to generate a ladder of molecular mass markers on Western blots of human skeletal muscle.

• Navarro C, Teijeira S, Garcia D
• [Limb-girdle dystrophy]
• Neurologia. 1999; 14: 369-70

• Galbiati F, Volonte D, Minetti C, Chu JB, Lisanti MP
• Phenotypic behavior of caveolin-3 mutations that cause autosomal dominant limb girdle muscular dystrophy (LGMD-1C). Retention of LGMD-1C caveolin-3 mutants within the golgi complex.
• J Biol Chem. 1999; 274: 25632-41
• Display abstract

• Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolae membrane domains in striated muscle cell types (cardiac and skeletal). Autosomal dominant limb girdle muscular dystrophy (LGMD-1C) in humans is due to mutations within the caveolin-3 gene: (i) a 9-base pair microdeletion that removes three amino acids within the caveolin scaffolding domain (DeltaTFT) or (ii) a missense mutation within the membrane spanning domain (P --> L). The molecular mechanisms by which these two mutations cause muscular dystrophy remain unknown. Here, we investigate the phenotypic behavior of these caveolin-3 mutations using heterologous expression. Wild type caveolin-3 or caveolin-3 mutants were transiently expressed in NIH 3T3 cells. LGMD-1C mutants of caveolin-3 (DeltaTFT or P --> L) were primarily retained at the level of a perinuclear compartment that we identified as the Golgi complex in double-labeling experiments, while wild type caveolin-3 was efficiently targeted to the plasma membrane. In accordance with these observations, caveolin-3 mutants formed oligomers of a much larger size than wild type caveolin-3 and were excluded from caveolae-enriched membrane fractions as seen by sucrose density gradient centrifugation. In addition, these caveolin-3 mutants were expressed at significantly lower levels and had a dramatically shortened half-life of approximately 45-60 min. However, caveolin-3 mutants were palmitoylated to the same extent as wild type caveolin-3, indicating that targeting to the plasma membrane is not required for palmitoylation of caveolin-3. In conclusion, we show that LGMD-1C mutations lead to formation of unstable high molecular mass aggregates of caveolin-3 that are retained within the Golgi complex and are not targeted to the plasma membrane. Consistent with its autosomal dominant form of genetic transmission, we demonstrate that LGMD-1C mutants of caveolin-3 behave in a dominant-negative fashion, causing the retention of wild type caveolin-3 at the level of the Golgi. These data provide a molecular explanation for why caveolin-3 levels are down-regulated in patients with this form of limb girdle muscular dystrophy (LGMD-1C).

• Bouju S et al.
• Exclusion of muscle specific actinin-associated LIM protein (ALP) gene from 4q35 facioscapulohumeral muscular dystrophy (FSHD) candidate genes.
• Neuromuscul Disord. 1999; 9: 3-10
• Display abstract

• Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder for which no candidate gene has yet been identified. The gene corresponding to one of the novel human cDNAs that we cloned on the basis of a muscle restricted expression pattern [Pietu G, Alibert O, Guichard B, et al. Genome Res 1996;6:492-503] was mapped in the region of the FSHD1A genetic locus, i.e. one of the loci involved in this muscular dystrophy. The corresponding encoded protein contains a PDZ and a LIM domain, two protein-protein interaction domains, and was very recently shown to bind alpha-actinin-2 and was named ALP (actinin-associated LIM protein) [Xia H, Winokur S, Kuo W, Altherr M, Bredt D. J Cell Biol 1997;139:507-515]. We raised a specific polyclonal anti-ALP serum against an ALP recombinant polypeptide to evaluate the size, level of expression and subcellular localization of ALP in three patients, clearly diagnosed with FSHD disease. Quantitative or qualitative alterations of ALP expression have not been detected in any of them, thus prompting us to exclude ALP as a FSHD gene candidate.

• Ono Y, Sorimachi H, Suzuki K
• New aspect of the research on limb-girdle muscular dystrophy 2A: a molecular biologic and biochemical approach to pathology.
• Trends Cardiovasc Med. 1999; 9: 114-8
• Display abstract

• p94, a muscle-specific member of the calpain family, also called calpain3 (CAPN3), has been identified as the gene product responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). To elucidate the molecular mechanism of LGMD2A, the effects of missense point mutations found in LGMD2A on the unique properties of p94 were studied. All of the mutants examined to date lose their proteolytic activity against fodrin, a cytoskeletal protein, strongly suggesting that of the specific properties of p94, the loss of protease activity is the prime cause of LGMD2A. Studies of LGMD2A and p94 suggest a novel molecular mechanism for muscular dystrophy, showing that a combined pathologic and biochemical approach is effective.

• McNally EM, Ly CT, Kunkel LM
• Human epsilon-sarcoglycan is highly related to alpha-sarcoglycan (adhalin), the limb girdle muscular dystrophy 2D gene.
• FEBS Lett. 1998; 422: 27-32
• Display abstract

• The dystrophin-glycoprotein complex (DGC) is critical for muscle membrane stability. The sarcoglycans are transmembrane proteins within the DGC, and the function of the sarcoglycans is unknown. Mutations in sarcoglycan genes cause autosomal recessive muscular dystrophy. We have identified a new sarcoglycan gene with high homology to alpha-sarcoglycan highlighting the redundancy of the DGC. This gene, named epsilon-sarcoglycan, has an identical intron-exon structure to alpha-sarcoglycan, and is more broadly expressed. The characterization of epsilon-sarcoglycan should make it possible to determine if it, like the other sarcoglycan genes, is mutated in muscular dystrophy.

• Liu J et al.
• Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular dystrophy.
• Nat Genet. 1998; 20: 31-6
• Display abstract

• Miyoshi myopathy (MM) is an adult onset, recessive inherited distal muscular dystrophy that we have mapped to human chromosome 2p13. We recently constructed a 3-Mb P1-derived artificial chromosome (PAC) contig spanning the MM candidate region. This clarified the order of genetic markers across the MM locus, provided five new polymorphic markers within it and narrowed the locus to approximately 2 Mb. Five skeletal muscle expressed sequence tags (ESTs) map in this region. We report that one of these is located in a novel, full-length 6.9-kb muscle cDNA, and we designate the corresponding protein 'dysferlin'. We describe nine mutations in the dysferlin gene in nine families; five are predicted to prevent dysferlin expression. Identical mutations in the dysferlin gene can produce more than one myopathy phenotype (MM, limb girdle dystrophy, distal myopathy with anterior tibial onset).

• Bashir R et al.
• A gene related to Caenorhabditis elegans spermatogenesis factor fer-1 is mutated in limb-girdle muscular dystrophy type 2B.
• Nat Genet. 1998; 20: 37-42
• Display abstract

• The limb-girdle muscular dystrophies are a genetically heterogeneous group of inherited progressive muscle disorders that affect mainly the proximal musculature, with evidence for at least three autosomal dominant and eight autosomal recessive loci. The latter mostly involve mutations in genes encoding components of the dystrophin-associated complex; another form is caused by mutations in the gene for the muscle-specific protease calpain 3. Using a positional cloning approach, we have identified the gene for a form of limb-girdle muscular dystrophy that we previously mapped to chromosome 2p13 (LGMD2B). This gene shows no homology to any known mammalian gene, but its predicted product is related to the C. elegans spermatogenesis factor fer-1. We have identified two homozygous frameshift mutations in this gene, resulting in muscular dystrophy of either proximal or distal onset in nine families. The proposed name 'dysferlin' combines the role of the gene in producing muscular dystrophy with its C. elegans homology.

• McNally EM et al.
• Caveolin-3 in muscular dystrophy.
• Hum Mol Genet. 1998; 7: 871-7
• Display abstract

• The dystrophin-glycoprotein complex (DGC) serves as a link between cytoplasmic actin, the membrane and the extracellular matrix of striated muscle. Genetic defects in genes encoding a subset of DGC proteins result in muscular dystrophy and a secondary decrease in other DGC proteins. Caveolae are dynamic structures that have been implicated in a number of functions including endocytosis, potocytosis and signal transduction. Caveolin (VIP-21) is thought to play a structural role in the formation of non-clathrin-coated vesicles in a number of different cell types. Caveolin-3, or M-caveolin, was identified as a muscle-specific form of the caveolin family. We show that caveolin-3 co-purifies with dystrophin, and that a fraction of caveolin-3 is a dystrophin-associated protein. We isolated the gene for human caveolin-3 and mapped it to chromosome 3p25. We determined the genomic organization of human caveolin-3 and devised a screening strategy to look for mutations in caveolin-3 in patients with muscular dystrophy. Of 82 patients screened, two nucleotide changes were found that resulted in amino acid substitutions (G55S and C71W); these changes were not seen in a control population. The amino acid changes map to a functionally important domain in caveolin-3, suggesting that these are not benign polymorphisms and instead are disease-causing mutations.

• Minetti C et al.
• Mutations in the caveolin-3 gene cause autosomal dominant limb-girdle muscular dystrophy.
• Nat Genet. 1998; 18: 365-8
• Display abstract

• Limb-girdle muscular dystrophy (LGMD) is a clinically and genetically heterogeneous group of myopathies, including autosomal dominant and recessive forms. To date, two autosomal dominant forms have been recognized: LGMD1A, linked to chromosome 5q, and LGMD1B, associated with cardiac defects and linked to chromosome 1q11-21. Here we describe eight patients from two different families with a new form of autosomal dominant LGMD, which we propose to call LGMD1C, associated with a severe deficiency of caveolin-3 in muscle fibres. Caveolin-3 (or M-caveolin) is the muscle-specific form of the caveolin protein family, which also includes caveolin-1 and -2. Caveolins are the principal protein components of caveolae (50-100 nm invaginations found in most cell types) which represent appendages or sub-compartments of plasma membranes. We localized the human caveolin-3 gene (CAV3) to chromosome 3p25 and identified two mutations in the gene: a missense mutation in the membrane-spanning region and a micro-deletion in the scaffolding domain. These mutations may interfere with caveolin-3 oligomerization and disrupt caveolae formation at the muscle cell plasma membrane.

• Liu J et al.
• Generation of a 3-Mb PAC contig spanning the Miyoshi myopathy/limb-girdle muscular dystrophy (MM/LGMD2B) locus on chromosome 2p13.
• Genomics. 1998; 49: 23-9
• Display abstract

• Miyoshi myopathy (MM) and limb-girdle muscular dystrophy subtype 2B (LGMD2B) map to the same region on chromosome 2p13. To facilitate the cloning of the defective gene causing these two diseases, we used a combination of chromosome walking and expressed sequence tag (EST) screening and identified 864 P1-derived artificial chromosomes (PACs) whose inserts map to the MM/LGMD2B candidate region and surrounding areas. Among them, 139 are from a chromosome 2-specific PAC library and 725 are from a total genomic PAC library. A 3-Mb contig spanning the candidate region for MM/LGMD2B was assembled. This contig contains 200 PACs, 10 known genetic markers, 5 new polymorphic markers, 57 sequence tagged sites (STSs) generated from PAC end fragments, and 4 random STSs. In addition, we mapped 24 ESTs to this contig and excluded 37 ESTs from the contig, thus eliminating them as candidate MM/LGMD2B genes. The high-resolution, sequence-ready PAC contig for the MM/LGMD2B region provides a backbone for the identification of the disease gene(s) and for clarification of the relationship between the two diseases.

• Kawai H et al.
• Clinical, pathological, and genetic features of limb-girdle muscular dystrophy type 2A with new calpain 3 gene mutations in seven patients from three Japanese families.
• Muscle Nerve. 1998; 21: 1493-501
• Display abstract

• We report on the clinical, pathological, and genetic features of 7 patients with limb-girdle muscular dystrophy type 2A (LGMD2A) from three Japanese families. The mean age of onset was 9.7+/-3.1 years (mean+/-SD), and loss of ambulance occurred at 38.5+/-2.1 years. Muscle atrophy was predominant in the pelvic and shoulder girdles, and proximal limb muscles. Muscle pathology revealed dystrophic changes. In two families, an identical G to C mutation at position 1080 the in calpain 3 gene was identified, and a frameshift mutation (1796insA) was found in the third family. The former mutation results in a W360R substitution in the proteolytic site of calpain 3, and the latter in a deletion of the Ca2+-binding domain.

• Bushby K, Anderson LV, Pollitt C, Naom I, Muntoni F, Bindoff L
• Abnormal merosin in adults. A new form of late onset muscular dystrophy not linked to chromosome 6q2.
• Brain. 1998; 121: 581-8
• Display abstract

• We have identified seven patients (including two sib pairs) with a predominantly late onset limb-girdle muscular dystrophy in whom an absence of merosin was noted on immunoblotting. Merosin immunocytochemistry was normal, and no abnormalities were detected on immunostaining for the various proteins known to be involved in the limb-girdle muscular dystrophies (alpha, beta, gamma, delta sarcoglycan and calpain 3). Apart from one patient, where muscle problems began in childhood, reported age at onset of muscle weakness involving initially the proximal muscles of the lower limbs ranged from 17 to 40 years. The pattern of muscle involvement was similar from patient to patient, with hypertrophy of at least the calf muscles, absence of scapular winging and predominant involvement of hip flexors and adductors and hamstrings more than quadriceps. Serum creatine kinase in all patients was at least 10 times normal, and muscle biopsies showed non-specific dystrophic features. We believe that the patients described here may represent a genetically distinct subset within the limb-girdle muscular dystrophy group.

• Anderson LV et al.
• Characterization of monoclonal antibodies to calpain 3 and protein expression in muscle from patients with limb-girdle muscular dystrophy type 2A.
• Am J Pathol. 1998; 153: 1169-79
• Display abstract

• Monoclonal antibodies were raised to two regions of calpain 3 (muscle-specific calcium-activated neutral protease), which is the product of the gene that is defective in limb-girdle muscular dystrophy type 2A. The antibodies produced characteristic patterns of bands on Western blots: normal calpain 3 protein was represented by bands at 94 kd, plus additional fragments at approximately 60 or 30 kd, according to the antibody used. Specificity was confirmed by the loss of all bands in patients with null gene mutations. The "normal" profile of bands was observed in muscle from 33 control subjects and 70 disease-control patients. Calpain 3 protein was found to be extremely stable in fresh human muscle, with full-size protein being detected 8 hours after the muscle had been removed. Blots of muscle from nine limb-girdle muscular dystrophy type 2A patients with defined mutations showed variation in protein expression, with seven showing a clear reduction in the abundance of protein detected. No simple relationship was found between the abundance and clinical severity. Two patients showed normal expression of the full-size 94 kd band accompanied by a clear reduction in the smaller fragments. This pattern was also observed in one patient with an undefined form of limb-girdle dystrophy. These results indicate that immunodiagnosis is feasible, but caution will need to be exercised with the interpretation of near-normal protein profiles.

• Richard I et al.
• Multiple independent molecular etiology for limb-girdle muscular dystrophy type 2A patients from various geographical origins.
• Am J Hum Genet. 1997; 60: 1128-38
• Display abstract

• Limb-girdle muscular dystrophies (LGMDs) are a group of neuromuscular diseases presenting great clinical heterogeneity. Mutations in CANP3, the gene encoding muscle-specific calpain, were used to identify this gene as the genetic site responsible for autosomal recessive LGMD type 2A (LGMD2A; MIM 253600). Analyses of the segregation of markers flanking the LGMD2A locus and a search for CANP3 mutations were performed for 21 LGMD2 pedigrees from various origins. In addition to the 16 mutations described previously, we report 19 novel mutations. These data indicate that muscular dystrophy caused by mutations in CANP3 are found in patients from all countries examined so far and further support the wide heterogeneity of molecular defects in this rare disease.

• Duggan DJ, Manchester D, Stears KP, Mathews DJ, Hart C, Hoffman EP
• Mutations in the delta-sarcoglycan gene are a rare cause of autosomal recessive limb-girdle muscular dystrophy (LGMD2).
• Neurogenetics. 1997; 1: 49-58
• Display abstract

• The dystrophin-based membrane cytoskeleton of muscle fibers has emerged as a critical multi-protein complex which seems to impart structural integrity on the muscle fiber plasma membrane. Deficiency of dystrophin causes the most common types of muscular dystrophy, Duchenne and Becker muscular dystrophies. Muscular dystrophy patients showing normal dystrophin protein and gene analysis are generally isolated cases with a presumed autosomal recessive inheritance pattern (limb-girdle muscular dystrophy). Recently, linkage and candidate gene analyses have shown that some cases of limb-girdle muscular dystrophy can be caused by deficiency of other components of the dystrophin membrane cytoskeleton. The most recently identified component, delta-sarcoglycan, has been found to show mutations in a series of Brazilian muscular dystrophy patients. All patients were homozygous for a protein-truncating carboxy-terminal mutation, and showed a deficiency of the four sarcoglycan proteins. To determine if delta-sarcoglycan deficiency occurred in other world populations, to identify the range of mutations and clinical phenotypes, and to test for the biochemical consequences of delta-sarcoglycan gene mutations, we studied Duchenne-like and limb-girdle muscular dystrophy patients who we had previously shown not to exhibit gene mutations of dystrophin, alpha-, beta-, or gamma-sarcoglycan for delta-sarcoglycan mutations (n = 54). We identified two American patients with novel nonsense mutations of delta-sarcoglycan (W30X, R165X). One was apparently homozygous, and we show likely consanguinity through homozygosity for 13 microsatellite loci covering a 38 cM region of chromosome 5. The second was heterozygous. Both were girls who showed clinical symptoms consistent with Duchenne muscular dystrophy in males. Our data shows that delta-sarcoglycan deficiency occurs in other world populations, and that most or all patients show a deficiency of the entire sarcoglycan complex, adding support to the hypothesis that these proteins function as a tetrameric unit.

• Hirabayashi K
• [Miyoshi distal muscular dystrophy]
• Nippon Rinsho. 1997; 55: 3190-4
• Display abstract

• Miyoshi distal muscular dystrophy (MDMD) is a young-adult-onset, autosomal recessive inherited dystrophy initially affecting the plantar flexers. We analysed 12 MDMD families, five with consanguineous marriage, for chromosomal linkage using polymorphic microsatellite DNA markers to map MDMD gene. A significant lod score was obtained with the 2p13 locus D2S291 (Zmax = 15.3 at theta = 0). A gene for autosomal recessive limb-girdle muscular dystrophy 2B (LGMD2B) was also mapped 2p13. The onset was in the late teens with weakness and wasting of the pelvic girdle muscles. Now we cannot exclude the possibility that the cause of these diseases are allelic variants in the same gene. YAC contig of the region was constructed. Scleening for muscle genes in the MDMD region is under way.

• Illarioshkin SN, Ivanova-Smolenskaya IA, Tanaka H, Poleshchuk VV, Markova ED, Tsuji S
• Refined genetic location of the chromosome 2p-linked progressive muscular dystrophy gene.
• Genomics. 1997; 42: 345-8
• Display abstract

• Autosomal recessive progressive muscular dystrophies may be clinically subclassified into limb-girdle muscular dystrophy (LGMD) and distal myopathy (DM), each clinical form being genetically heterogeneous. Genes for LGMD type 2B and Miyoshi myopathy (a form of DM) have been mapped to essentially the same region on chromosome 2p. We described recently a large inbred family with autosomal recessive muscular dystrophy in which the LGMD and the DM phenotypes were manifested in separate affected members, and we assigned the gene for this condition to the same locus as in LGMD2B and Miyoshi myopathy. Here we report extended haplotypes in this family generated from 15 markers located at the region of interest on chromosome 2p13. Key recombinants allowed us to reduce further the candidate region for this polymorphic condition and defined the loci D2S327 and D2S2111 as the most likely boundaries of the mutant gene.

• Illarioshkin SN et al.
• [Mapping of the gene for autosomal-recessive progressive muscular dystrophy in an isolate from a highland region of Dagestan to chromosome 2-13]
• Genetika. 1997; 33: 1551-8
• Display abstract

• A unique inbred Avar family from an isolate of the Dagestan highland was studied. Unusual phenotypic expression of autosomal recessive progressive muscular dystrophy was revealed in 12 members of this family from three generations. Limb-girdle (proximal) muscular dystrophy (LGMD) was detected in nine patients, while the other three patients displayed typical distal myopathy (DM). Genetic linkage analysis with several candidate loci determining various forms of muscular dystrophy allowed a gene for this polymorphic syndrome to be assigned to chromosome 2p13. In spite of the difference in clinical manifestation, all patients appeared to be homozygous for a unique haplotype. This implies the founder effect and proves the same genetic basis of LGMD and DM in the family. Recombination analysis showed that the centromeric and telomeric ends of the gene region are marked with D2S2111 and D2S327, respectively (genetic distance < 1 cM). This region is overlapped by two larger regions in which the genes for LGMD type 2B (LGMD2B) and Miyoshi myopathy were recently mapped. Complex analysis of clinical and genetic data indicated that LGMD2B, Miyoshi myopathy, and the revealed polymorphic syndrome may represent allelic variants of 2p13-linked autosomal recessive muscular dystrophy.

• Bashir R et al.
• Genetic and physical mapping at the limb-girdle muscular dystrophy locus (LGMD2B) on chromosome 2p.
• Genomics. 1996; 33: 46-52
• Display abstract

• The limb-girdle muscular dystrophies (LGMD) are a genetically heterogeneous group of disorders, different forms of which have been mapped to at least six distinct genetic loci. We have mapped an autosomal recessive form of LGMD (LGMD2B) to chromosome 2p13. Two other conditions have been shown to map to this region or to the homologous region in mouse: a gene for a form of autosomal recessive distal muscular dystrophy, Miyoshi myopathy, shows linkage to the same markers on chromosome 2p as LGMD2B, and an autosomal recessive mouse mutation mnd2, in which there is rapidly progressive paralysis and muscle atrophy, has been mapped to mouse chromosome 6 to a region showing conserved synteny with human chromosome 2p12-p13. We have assembled a 6-cM YAC contig spanning the LGMD2B locus and have mapped seven genes and 13 anonymous polymorphic microsatellites to it. Using haplotype analysis in the linked families, we have narrowed our region of interest to a 0-cM interval between D2S2113 and D2S2112/D2S145, which does not overlap with the critical region for mnd2 in mouse. Use of these most closely linked markers will help to determine the relationship between LGMD2B and Miyoshi myopathy. YACs selected from our contig will be the starting point for the cloning of the LGMD2B gene and thereby establish the biological basis for this form of muscular dystrophy and its relationship with the other limb-girdle muscular dystrophies.

• McNally EM et al.
• Mutations that disrupt the carboxyl-terminus of gamma-sarcoglycan cause muscular dystrophy.
• Hum Mol Genet. 1996; 5: 1841-7
• Display abstract

• Recently, mutations in the genes encoding several of the dystrophin-associated proteins have been identified that produce phenotypes ranging from severe Duchenne-like autosomal recessive muscular dystrophy to the milder limb-girdle muscular dystrophies (LGMDs). LGMD type 2C is generally associated with a more severe clinical course and is prevalent in northern Africa. A previous study identified a single base pair deletion in the gene encoding the dystrophin-associated protein gamma-sarcoglycan in a number of Tunisian muscular dystrophy patients. To investigate whether gamma-sarcoglycan gene mutations cause autosomal recessive muscular dystrophy in other populations, we studied 50 muscular dystrophy patients from the United States and Italy. The muscle biopsies from these 50 patients showed no abnormality of dystrophin but did show diminished immunostaining for the dystrophin-associated protein alpha-sarcoglycan. Four patients with a severe muscular dystrophy phenotype were identified with homozygous, frameshifting mutations in gamma-sarcoglycan. Two of the four have microdeletions that disrupt the distal carboxyl-terminus of gamma-sarcoglycan yet result in a complete absence of gamma-and beta-sarcoglycan suggesting the importance of this region for stability of the sarcoglycan complex. This region of gamma-sarcoglycan, like beta-sarcoglycan, has a number of cysteine residues similar to those in epidermal growth factor cysteine-rich regions.

• Richard I, Beckmann JS
• Molecular cloning of mouse canp3, the gene associated with limb-girdle muscular dystrophy 2A in human.
• Mamm Genome. 1996; 7: 377-9

• Allamand V et al.
• Preferential localization of the limb-girdle muscular dystrophy type 2A gene in the proximal part of a 1-cM 15q15.1-q15.3 interval.
• Am J Hum Genet. 1995; 56: 1417-30
• Display abstract

• A gene for a recessive form of limb-girdle muscular dystrophy (LGMD2A) has been localized to chromosome 15. A physical map of the 7-cM candidate 15q15.1-q21.1 region has been constructed by means of a 10-12-Mb continuum of overlapping YAC clones. New microsatellite markers developed from these YACs were genotyped on large, consanguineous LGMD2A pedigrees from different origins. The identification of recombination events in these families allowed the restriction of the LGMD2A region to an estimated 1-cM interval, equivalent to approximately 3-4 Mb. Linkage disequilibrium data on genetic isolates from the island of Reunion and from the Amish community suggest a preferential location of the LGMD2A gene in the proximal part of this region. Analysis of the interrelated pedigrees from Reunion revealed the existence of at least six different carrier haplotypes. This allelic heterogeneity is incompatible with the presumed existence of a founder effect and suggests that multiple LGMD2A mutations may segregate in this population.

• Campbell KP
• Adhalin gene mutations and autosomal recessive limb-girdle muscular dystrophy.
• Ann Neurol. 1995; 38: 353-4

• Chiannilkulchai N, Pasturaud P, Richard I, Auffray C, Beckmann JS
• A primary expression map of the chromosome 15q15 region containing the recessive form of limb-girdle muscular dystrophy (LGMD2A) gene.
• Hum Mol Genet. 1995; 4: 717-25
• Display abstract

• Previous genetic and physical studies of LGMD2A, an autosomal recessive form of limb-girdle muscular dystrophy, have led to the establishment of a 10-12 Mb YAC contig encompassing the morbid locus. In order to progress toward the identification of the gene involved in LGMD2A, a primary transcription map of this genomic region was generated. The direct cDNA selection strategy was used with three YACs covering the candidate region and two different muscle cDNA libraries. Seventeen transcription units were identified among 171 cDNA fragments analysed. Five sequences corresponded to known genes, and twelve to new ones. They were characterized for their sequences, physical positions within the YAC contig, and expression patterns. Among those specifically transcribed in muscle, the calpain gene is a good positional and functional candidate for LGMD2A.

• Richard I, Roudaut C, Fougerousse F, Chiannilkulchai N, Beckmann JS
• An STS map of the limb girdle muscular dystrophy type 2A region.
• Mamm Genome. 1995; 6: 754-6

• Sorimachi H et al.
• Muscle-specific calpain, p94, responsible for limb girdle muscular dystrophy type 2A, associates with connectin through IS2, a p94-specific sequence.
• J Biol Chem. 1995; 270: 31158-62
• Display abstract

• p94, a muscle-specific member of calpain family, is unique in that it undergoes rapid and exhaustive autolysis with a half-life of less than 1 h resulting in its disappearance from muscle. Recently, p94 was shown to be responsible for limb girdle muscular dystrophy type 2A. To elucidate the muscular proteolytic system mediated by p94 and to solve the mystery of its unusually rapid autolysis, we searched for p94-binding proteins by the two-hybrid system. Although calpain small subunit plays a crucial role for regulation of ubiquitous calpains, it did not associate with p94. After a screening of skeletal muscle library, connectin (or titin), a gigantic filamentous protein spanning the M- to Z-lines of muscle sarcomere, was found to bind to p94 through a p94-specific region, IS2. The connectin-insoluble fraction of washed myofibrils contained full-length intact p94, suggesting that connectin regulates p94 activity.

• Parrish JE, Ciccodicola A, Wehhert M, Cox GF, Chen E, Nelson DL
• A muscle-specific DNase I-like gene in human Xq28.
• Hum Mol Genet. 1995; 4: 1557-64
• Display abstract

• A novel cDNA which maps to human Xq28 has been isolated and characterized. Sequence similarity to DNase I is high at the DNA and peptide sequence levels. The transcript is present at highest levels in skeletal and cardiac muscle, with lower expression in other tissues. Mutation analysis has been performed using DNA samples from two unrelated patients with Barth syndrome, and from 11 unrelated patients with Emery-Dreifuss muscular dystrophy, two genetic disorders linked to Xq28. No disease-associated mutations were detected in the coding region of the gene; however, a novel 190 base pair insertion/deletion polymorphism was found in the 3' untranslated region. Translation of the long open reading frame found in the cDNA yields a putative 302 amino acid protein with 37.6% identity to human DNase I. The protein is predicted to contain a signal sequence at the amino terminus, a transmembrane domain near the carboxyl terminus, and a helix-loop-helix domain.

• Fougerousse F et al.
• Mapping of a chromosome 15 region involved in limb girdle muscular dystrophy.
• Hum Mol Genet. 1994; 3: 285-93
• Display abstract

• A gene responsible for an autosomal recessive form of limb girdle muscular dystrophy (LGMD2, MIM number 253600) has been localized on chromosome 15. After genotyping additional markers of this chromosome, two were found to flank the disease locus within an interval that was assessed as 7 centiMorgans. The screening of the CEPH YAC libraries with the corresponding probes allowed the isolation of YACs which were used in fluorescence in situ hybridization to define the LGMD2 cytogenetic interval as 15q15.1-15q21.1. Four different approaches were pursued for the establishment of the physical map of this area which allowed the assembly of an uninterrupted YAC contig spanning an estimated 10-12 megabases, with an average STS resolution of 140 kb or for the 25 polymorphic microsatellites on this map, of 400 kb. Twelve genes and 25 genetic markers were positioned in this contig, which is constituted of a minimum of 10 clones.

• Velinov M et al.
• Limb-girdle muscular dystrophy is closely linked to the fibrillin locus on chromosome 15.
• Connect Tissue Res. 1993; 29: 13-21
• Display abstract

• Limb-girdle Muscular Dystrophy (LGMD) is a rare form of muscular dystrophy inherited as an autosomal recessive trait. The LGMD locus was recently mapped to chromosome 15. We tested the hypothesis that fibrillin is a candidate in the etiology of the disorder by genetic linkage analysis. A large Amish kindred segregating the disorder was genotyped for two markers specific for the fibrillin gene on chromosome 15. A total of 105 individuals were genotyped and a maximum LOD score of Z = 9. 135 at theta = 0.04 was obtained. Our results confirmed the mapping of the LGMD on chromosome 15 and excluded fibrillin as a candidate molecule. These data will be useful in the construction of a fine map of the region surrounding the LGMD locus, a prerequisite for the cloning of the LGMD gene.

• Wijmenga C, Hofker MH, Padberg GW, Frants RR
• Genetic mapping of facioscapulohumeral muscular dystrophy.
• Mol Cell Biol Hum Dis Ser. 1993; 3: 111-38

• Richard I et al.
• Mapping of the formin gene and exclusion as a candidate gene for the autosomal recessive form of limb-girdle muscular dystrophy.
• Hum Mol Genet. 1992; 1: 621-4
• Display abstract

• Limb-Girdle Muscular Dystrophy (LGMD) is a myopathy with clinical and transmission heterogeneity. The recessive form, LGMD2, has been recently mapped by linkage analysis to 15q. As an attempt to identify the gene involved in this pathology, we tested as candidate gene the LD locus, called LD for limb deformity. This gene has recently been identified and mapped to chromosome 15q13-q14. It is homologous to the murine formin gene which is localized to mouse chromosome 2. Mutations in this murine gene have been shown to cause limb deformity and kidney defect. YAC clones containing the LD gene were isolated and utilised to confirm the cytogenetic localisation. Internal DNA polymorphisms of the LD locus were analyzed in LGMD2 and CEPH families. The LD gene was mapped between the alpha cardiac actin gene and the D15S24 locus. Crossovers between the LGMD2 and the LD loci excluded the LD gene as a candidate for LGMD2.

• Speer MC et al.
• Confirmation of genetic heterogeneity in limb-girdle muscular dystrophy: linkage of an autosomal dominant form to chromosome 5q.
• Am J Hum Genet. 1992; 50: 1211-7
• Display abstract

• Limb-girdle muscular dystrophy (LGMD) is a clinically and genetically heterogenous group of disorders, with both recessive and dominant forms reported. Recently, a series of recessive LGMD families were linked to chromosome 15q. We report herein the results of our linkage studies in a previously reported large autosomal dominant family. The LGMD gene in this family was localized to chromosome 5q22.3-31.3 by using a series of CA(n) microsatellite repeat markers. Linkage to 15q was excluded. These findings confirm genetic heterogeneity in this clinically diverse syndrome.

• Beckmann JS et al.
• A gene for limb-girdle muscular dystrophy maps to chromosome 15 by linkage.
• C R Acad Sci III. 1991; 312: 141-8
• Display abstract

• Limb-girdle muscular dystrophy (LGMD) is inherited as a monogenic, autosomal recessive trait. A genetically homogeneous group of families from the Isle of La Reunion, comprising individuals at high risk for this disorder, was systematically analysed using a panel of 85 polymorphic markers spanning approximately 30% of the human genome. Linkage was detected between the LGMD gene and the marker D15S25, uncovered with the probe pTHH114 and restriction enzyme RsaI (lod score = 5.52 at a 0 = 0.0), localising this gene onto chromosome 15. Such a lod score corresponds to odds of 3.3 x 105 in favor of linkage versus absence of linkage. Additional families from other populations will need to be examined before the role of this newly identified locus can be understood.

• Tsukahara T, Ishiura S, Arahata K, Sugita H
• [Muscular dystrophy]
• Tanpakushitsu Kakusan Koso. 1990; 35: 1246-53

• Ishiura S, Arahata K, Tsukahara T, Sugita H
• [Dystrophin deficiency in Duchenne muscular dystrophy]
• Seikagaku. 1989; 61: 1371-5

• Oexle K, Schliwa M
• Dystrophin under scrutiny.
• Biol Cell. 1989; 67: 239-41

• Slater CR
• Muscle proteins and muscular dystrophy.
• Curr Opin Cell Biol. 1989; 1: 110-4

• Hoffman EP, Kunkel LM
• Dystrophin abnormalities in Duchenne/Becker muscular dystrophy.
• Neuron. 1989; 2: 1019-29

• Love DR, Davies KE
• Duchenne muscular dystrophy: the gene and the protein.
• Mol Biol Med. 1989; 6: 7-17

• Worton RG, Thompson MW
• Genetics of Duchenne muscular dystrophy.
• Annu Rev Genet. 1988; 22: 601-29

• Sorensen PS, Sorensen T, Sorensen KV
• Reversible limb-girdle muscular dystrophy.
• Lancet. 1988; 2: 913-913

• Wood DS et al.
• Is nebulin the defective gene product in Duchenne muscular dystrophy?
• N Engl J Med. 1987; 316: 107-8

• Yates JR, Emery AE
• A population study of adult onset limb-girdle muscular dystrophy.
• J Med Genet. 1985; 22: 250-7
• Display abstract

• Complete ascertainment of adult onset limb-girdle muscular dystrophy in the Lothian Region of Scotland was attempted. Ten index cases were identified giving a prevalence of 1.3 per 100 000 (0.9 per 100 000 for cases where the diagnosis of muscular dystrophy was supported by both electromyographic and muscle biopsy findings). In these 10 sibships there had been 11 affected subjects, significantly less than the 16.5 cases expected for autosomal recessive inheritance. Excluding cases suspected of being Becker muscular dystrophy, the prevalence was 0.7 per 100 000 (0.3 per 100 000 for proven cases of muscular dystrophy) and there remained a significant difference between the number of cases observed (5) and the number expected (9.1) for autosomal recessive inheritance. The prevalence of limb-girdle muscular dystrophy with onset in adult life has apparently declined over the past 30 years, as would be expected with the recognition of other conditions which cause the same pattern of weakness, making this a relatively rare disorder which should only be considered when other diagnoses have been excluded. The possibility that some cases diagnosed as limb-girdle muscular dystrophy may have had Becker muscular dystrophy emphasises the urgent need for a greater understanding of the biochemical basis of these conditions so that such diagnostic and genetic counselling dilemmas can be resolved.

• Perry SV
• Duchenne muscular dystrophy.
• Biochem Soc Trans. 1984; 12: 362-5

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