Limb girdle muscular dystrophy in a sibling pair with a homozygous Ser606Leu mutation in the alternatively spliced IS2 region of calpain 3

Previous family studies revealed a large number of calpain 3 ( CAPN3 ) mutations that cause recessive forms of limb girdle muscular dystrophy (LGMD2A) with selective atrophy of the proximal limb muscles. Correlations between the nature and site of a particular mutation and its corresponding phenotype, however, can only be established from homozygous mutations, which are particularly rare in the alternatively spliced NS, IS1 and IS2 regions of CAPN3. Here we identified a sibling pair with LGMD2A-type muscular dystrophy caused by a homozygous Ser606Leu (S606L) substitution in the IS2 linker domain. Normal protein levels, unaltered myofibrillar targeting and conserved calcium-induced autocatalytic activity of the mutated protein could be demonstrated in muscle biopsies from one patient. Despite this inconspicuous modification of the IS2 linker between domains III and IV, both patients developed signs and symptoms of the disease within their second decade of life. The unexpected severity of the clinical manifestation points to the high relevance of the calpain 3-specific IS2 segment between domains III and IV. We conclude that the structural motif around the Ser606 residue represents an important functional site that may regulate the transient activation and limited proteolysis of calpain 3.     

Molecular and cellular basis of calpainopathy (limb girdle muscular dystrophy type 2A)

Limb girdle muscular dystrophy type 2A results from mutations in the gene encoding the calpain 3 protease. Mutations in this disease are inherited in an autosomal recessive fashion and result in progressive proximal skeletal muscle wasting but no cardiac abnormalities. Calpain 3 has been shown to proteolytically cleave a wide variety of cytoskeletal and myofibrillar proteins and to act upstream of the ubiquitin-proteasome pathway. In this review, we summarize the known biochemical and physiological features of calpain 3 and hypothesize why mutations result in disease.     

Ins and outs of therapy in limb girdle muscular dystrophies

Muscular dystrophies are hereditary degenerative muscle diseases that cause life-long disability in patients. They comprise the well-known Duchenne Muscular Dystrophy (DMD) but also the group of Limb Girdle Muscular Dystrophies (LGMD) which account for a third to a fourth of DMD cases. From the clinical point of view, LGMD are characterised by predominant effects on the proximal limb muscles. The LGMD group is still growing today and consists of 19 autosomal dominant and recessive forms (LGMD1A to LGMD1G and LGMD2A to LGMD2M). The proteins involved are very diverse and include sarcomeric, sarcolemmal and enzymatic proteins. With respect to this variability and in line with the intense search for a potent therapeutic approach for DMD, many different strategies have been tested in rodent models. These include replacing the lost function by gene transfer or stem cell transplantation, using a related protein for functional substitution, increasing muscle mass, or blocking the molecular pathological mechanisms by pharmacological means to alleviate the symptoms. The purpose of this review is to summarize current data arising from these preclinical studies and to examine the potential of the tested strategies to lead to clinical applications.     

TIMP3 mutation in Sorsby's fundus dystrophy: molecular insights

Sorsby's fundus dystrophy (SFD) is a rare autosomal dominant disorder that results in degeneration of the macular region of the retina, with onset usually in the fourth to fifth decade of life. It leads to the rapid loss of central vision, often followed by further loss of peripheral vision. SFD shares several pathological features commonly found in the 'wet' or exudative form of age-related macular degeneration (AMD), the most common cause of blindness in the elderly in developed countries. These phenotypic similarities have led to SFD being proposed as an acceptable genetic model for AMD. Whereas AMD appears to have a complex aetiology, with both genetic and environmental factors playing a role, SFD has been shown to be a single-gene disorder, linked to mutations in exon 5 of the tissue inhibitor of metalloproteinases 3 (TIMP3) gene on chromosome 22q12-q13. This review confines itself to a discussion of the known biochemical properties of the wild-type and SFD TIMP3 proteins and attempts to relate these to the pathology encountered in SFD patients. We also discuss briefly how, despite the lack of inherited mutations in the structural gene, the TIMP3 protein might play a role in the onset and progression of AMD.     

Solution structure of the inner DysF domain of myoferlin and implications for limb girdle muscular dystrophy type 2b

Mutations in the protein dysferlin, a member of the ferlin family, lead to limb girdle muscular dystrophy type 2B and Myoshi myopathy. The ferlins are large proteins characterised by multiple C2 domains and a single C-terminal membrane-spanning helix. However, there is sequence conservation in some of the ferlin family in regions outside the C2 domains. In one annotation of the domain structure of these proteins, an unusual internal duplication event has been noted where a putative domain is inserted in between the N- and C-terminal parts of a homologous domain. This domain is known as the DysF domain. Here, we present the solution structure of the inner DysF domain of the dysferlin paralogue myoferlin, which has a unique fold held together by stacking of arginine and tryptophans, mutations that lead to clinical disease in dysferlin.     

Limb-girdle muscular dystrophy type 2H associated with mutation in TRIM32, a putative E3-ubiquitin-ligase gene

Limb-girdle muscular dystrophy type 2H (LGMD2H) is a mild autosomal recessive myopathy that was first described in the Manitoba Hutterite population. Previous studies in our laboratory mapped the causative gene for this disease to a 6.5-Mb region in chromosomal region 9q31-33, flanked by D9S302 and D9S1850. We have now used additional families and a panel of 26 microsatellite markers to construct haplotypes. Twelve recombination events that reduced the size of the candidate region to 560 kb were identified or inferred. This region is flanked by D9S1126 and D9S737 and contains at least four genes. Exons of these genes were sequenced in one affected individual, and four sequence variations were identified. The families included in our study and 100 control individuals were tested for these variations. On the basis of our results, the mutation in the tripartite-motif-containing gene (TRIM32) that replaces aspartate with asparagine at position 487 appears to be the causative mutation of LGMD2H. All affected individuals were found to be homozygous for D487N, and this mutation was not found in any of the controls. This mutation occurs in an NHL (named after the proteins NCL1, HT2A, and LIN-41) domain at a position that is highly conserved. NHL domains are known to be involved in protein-protein interactions. Although the function of TRIM32 is unknown, current knowledge of the domain structure of this protein suggests that it may be an E3-ubiquitin ligase. If proven, this represents a new pathogenic mechanism leading to muscular dystrophy.     

Utility of MLPA in mutation analysis and carrier detection for Duchenne muscular dystrophy

CONTEXT:

Multiplex ligation probe amplification (MLPA) is a new technique to identify deletions and duplications and can evaluate all 79 exons in dystrophin gene in patients with Duchenne muscular dystrophy (DMD). Being semi-quantitative, MLPA is also effective in detecting duplications and carrier testing of females; both of which cannot be done using multiplex PCR. It has found applications in diagnostics of many genetic disorders.

AIM:

To study the utility of MLPA in diagnosis and carrier detection for DMD.

MATERIALS AND METHODS:

Mutation analysis and carrier detection was done by multiplex PCR and MLPA and the results were compared.

RESULTS AND CONCLUSIONS:

We present data showing utility of MLPA in identifying mutations in cases with DMD/BMD. In the present study using MLPA, we identified mutations in additional 5.6% cases of DMD in whom multiplex PCR was not able to detect intragenic deletions. In addition, MLPA also correctly confirmed carrier status of two obligate carriers and revealed carrier status in 6 of 8 mothers of sporadic cases.     

Localization of calpain 3 in human skeletal muscle and its alteration in limb-girdle muscular dystrophy 2A muscle

Calpain 3/p94, the skeletal muscle-specific isoform of the calpain large subunit family, is a protein product of the gene responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). Through yeast two-hybrid experiments, calpain 3 has been shown to bind to titin in myofibrils [Sorimachi et al. (1995) J. Biol. Chem. 270, 31158-31162]. However, because of extensive autolysis activity, calpain 3 localization in skeletal muscle has been undefined. In this study, we generated a polyclonal antibody against an N-terminal 98-amino-acid calpain 3 fragment, which is not homologous to the corresponding regions of other conventional calpains. This antibody stained myofibrils with a unique repeated doublet-pattern. Confocal microscopic observation with marker antibodies confirmed that calpain 3 is localized in the N2 region of myofibrils. Furthermore, using this antibody, we examined the localization of calpain 3 in LGMD2A muscles.     

Mild and severe muscular dystrophy caused by a single gamma-sarcoglycan mutation.

Autosomal recessive muscular dystrophy is genetically heterogeneous. One form of this disorder, limb-girdle muscular dystrophy type 2C (LGMD 2C), is prevalent in northern Africa and has been shown to be associated with a single mutation in the gene encoding the dystrophin-associated protein gamma-sarcoglycan. The previous mutation analysis of gamma-sarcoglycan required the availability of muscle biopsies. To establish a mutation assay for genomic DNA, the intron-exon structure of the gamma-sarcoglycan gene was determined, and primers were designed to amplify each of the exons encoding gamma-sarcoglycan. We studied a group of Brazilian muscular dystrophy patients for mutations in the gamma-sarcoglycan gene. These patients were selected on the basis of autosomal inheritance and/or the presence of normal dystrophin and/or deficiency of alpha-sarcoglycan immunostaining. Four of 19 patients surveyed had a single, homozygous mutation in the gamma-sarcoglycan gene. The mutation identified in these patients, all of African-Brazilian descent, is identical to that seen in the North African population, suggesting that even patients of remote African descent may carry this mutation. The phenotype in these patients varied considerably. Of four families with an identical mutation, three have a severe Duchenne-like muscular dystrophy. However, one family has much milder symptoms, suggesting that other loci may be present that modify the severity of the clinical course resulting from gamma-sarcoglycan gene mutations.     

Caveolae and caveolin-3 in muscular dystrophy

Caveolae are vesicular invaginations of the plasma membrane, and function as 'message centers' for regulating signal transduction events. Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolar membrane domains in skeletal muscle and in the heart. Several mutations within the coding sequence of the human caveolin-3 gene (located at 3p25) have been identified. Mutations that lead to a loss of approximately 95% of caveolin-3 protein expression are responsible for a novel autosomal dominant form of limb-girdle muscular dystrophy (LGMD-1C) in humans. By contrast, upregulation of the caveolin-3 protein is associated with Duchenne muscular dystrophy (DMD). Thus, tight regulation of caveolin-3 appears essential for maintaining normal muscle health and homeostasis.     

A novel emerin mutation in a Japanese patient with Emery-Dreifuss muscular dystrophy

Sequencing of the STA gene in a patient with Emery-Dreifuss muscular dystrophy showed a 1-bp deletion of C at nucleotide 672 or 673. This deletion causes a frameshift, changing the amino acid sequence (amino acids 206–235) and generating an early stop codon. Received: 21 September 1995     

Trim32 is a ubiquitin ligase mutated in limb girdle muscular dystrophy type 2H that binds to skeletal muscle myosin and ubiquitinates actin

Trim32 belongs to the tripartite motif (TRIM) protein family, which is characterized by a common domain structure composed of a RING-finger, a B-box, and a coiled-coil motif. In addition to these motifs, Trim32 possesses six C-terminal NHL-domains. A point mutation in one NHL domain (D487N) has been linked to two forms of muscular dystrophy called limb girdle muscular dystrophy type 2H and sarcotubular myopathy. In the present study we demonstrate that Trim32 is an E3 ubiquitin ligase that acts in conjunction with ubiquitin-conjugating enzymes UbcH5a, UbcH5c, and UbcH6. Western blot analysis showed that Trim32 is expressed primarily in skeletal muscle, and revealed its differential expression from one muscle to another. The level of Trim32 expression was elevated significantly in muscle undergoing remodeling due to changes in weight bearing. Furthermore, expression of Trim32 was induced in myogenic differentiation. Thus, variability in Trim32 expression in different skeletal muscles could be due to induction of Trim32 expression upon changes in physiological conditions. We show that Trim32 associates with skeletal muscle thick filaments, interacting directly with the head and neck region of myosin. Our data indicate that myosin is not a substrate of Trim32; however, Trim32 was found to ubiquitinate actin in vitro and to cause a decrease in the level of endogenous actin when transfected into HEK293 cells. In conclusion, our results demonstrate that Trim32 is a ubiquitin ligase that is expressed in skeletal muscle, can be induced upon muscle unloading and reloading, associates with myofibrils and is able to ubiquitinate actin, suggesting its likely participation in myofibrillar protein turnover, especially during muscle adaptation.     

Molecular dissection of TIMP3 mutation S156C associated with Sorsby fundus dystrophy

Sorsby fundus dystrophy (SFD) is an autosomal dominant macular degeneration of late onset. A key feature of the disease is the thickening of Bruch's membrane, an ECM structure located between the RPE and the choroid. SFD is caused by mutations in the gene encoding the ECM-associated tissue inhibitor of metalloproteases-3 (TIMP3). We have recently generated two Timp3 gene-targeted mouse lines, one deficient for the murine gene (Timp3-/-) and one carrying an SFD-related S156C mutation. Based on extracts and cell cultures derived from tissues of these animals we now evaluated TIMP3 functionality and its contribution to SFD. We show that the activity levels of TIMP3 target proteases including TACE, ADAMTS4/5 and aggrecan-cleaving MMPs are similar in Timp3S156/+ and Timp3S156C/S156C mice when compared to controls. In Timp3-/- mice, a significant enhancement of enzyme activity was observed for TACE but not for ADAMTS4/5 and MMPs indicating a compensatory effect of other inhibitors regulating the latter two groups of proteases. Fibrin bead assays show that angiogenesis in Timp3S156/+ and Timp3S156C/S156C mice is not altered whereas increased formation of capillary tubes was observed in Timp3-/- animals over controls. Rescue experiments using recombinant proteins demonstrate that the inhibitory activities of TIMP3 towards TACE and aggrecan-cleaving MMPs as well as the anti-angiogenic properties of TIMP3 are not impaired by SFD mutation S156C. We finally demonstrate that wild-type and S156C-TIMP3 proteins block the binding of VEGF to its receptor VEGFR2 to a similar extent. Taken together, this study shows that S156C-TIMP3 retains its known functional properties suggesting that causes other than an imbalance in protease or angiogenic activities represent the primary molecular defect underlying SFD.     

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.

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).     

Evidence for mutation by unequal sister chromatid exchange in the Duchenne muscular dystrophy gene.

We have studied three families each containing a male with Duchenne or Becker muscular dystrophy. Southern blot analysis using both genomic and cDNA probes revealed that an exon-containing segment of DNA within the gene is duplicated in the probands, their mothers, and, in two cases, their sisters. The grandpaternal origin of the duplication has been demonstrated in these families by RFLP and duplication analysis. The results suggest that unequal sister-chromatid exchange, which most likely occurred in the germ cell lineage of the proband's grandfather, is responsible for generating these duplications and that this type of intrachromosomal rearrangement, although rarely reported in humans, is not uncommon in the muscular dystrophy gene.     

Loss of calpain 3 proteolytic activity leads to muscular dystrophy and to apoptosis-associated IkappaBalpha/nuclear factor kappaB pathway perturbation in mice

Calpain 3 is known as the skeletal muscle-specific member of the calpains, a family of intracellular nonlysosomal cysteine proteases. It was previously shown that defects in the human calpain 3 gene are responsible for limb girdle muscular dystrophy type 2A (LGMD2A), an inherited disease affecting predominantly the proximal limb muscles. To better understand the function of calpain 3 and the pathophysiological mechanisms of LGMD2A and also to develop an adequate model for therapy research, we generated capn3-deficient mice by gene targeting. capn3-deficient mice are fully fertile and viable. Allele transmission in intercross progeny demonstrated a statistically significant departure from Mendel's law. capn3-deficient mice show a mild progressive muscular dystrophy that affects a specific group of muscles. The age of appearance of myopathic features varies with the genetic background, suggesting the involvement of modifier genes. Affected muscles manifest a similar apoptosis-associated perturbation of the IkappaBalpha/nuclear factor kappaB pathway as seen in LGMD2A patients. In addition, Evans blue staining of muscle fibers reveals that the pathological process due to calpain 3 deficiency is associated with membrane alterations.