Preferential Localization of the Limb-Girdle Muscular Dystrophy Type 2A Gene in the Proximal Part of a 1-cM 15q15.1-q15.3 Interval

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 ~3–4 Mb. Linkage disequilibrium data on genetic isolates from the island of Réunion 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 Réunion 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.     

Klinik und Genetik der Gliedergürteldystrophien

Limb girdle muscular dystrophies (LGMDs) are a clinically and genetically heterogeneous group of muscle disorders. Seven dominant (LGMD1A through G) and 15 recessive forms (LGMD2A through O) have been described. They often start in adolescence, and most patients end up wheelchair-bound 2–4 decades later. The syndrome begins in the pelvic girdle. Muscles of the shoulder girdle follow after a variable time interval. Allelic variants may present with a distal predilection of muscles, such as Miyoshi myopathy, which is caused by mutations in the dysferlin gene. The most frequent types of LGMD are calpainopathies (LGMD1A), mutations in the FKRP gene (LGMD2i), and dysferlinopathies (LGMD2B). Sarcoglycanopathies, which often start in childhood, are the next most frequent forms. In many LGMDs, a sarcolemmal protein is affected. Because of the huge heterogeneity, molecular genetic analysis normally follows a muscle biopsy and includes an extensive immunohistochemical workup. A specific therapy for this group of diseases is not yet available. Human genetic counseling is of primary importance. Treatment of contractures as well as special care for a developing cardiomyopathy are helpful for the patient.     

Evidence for Locus Heterogeneity in Autosomal Dominant Limb-Girdle Muscular Dystrophy

Limb-girdle muscular dystrophy (LGMD) is a diagnostic classification encompassing a broad group of proximal myopathies. A gene for the dominant form of LGMD (LGMD1A) has recently been localized to a 7-cM region of chromosome 5q between D5S178 and IL9. We studied three additional dominant LGMD families for linkage to these two markers and excluded all from localization to this region, providing evidence for locus heterogeneity within the dominant form of LGMD. Although the patterns of muscle weakness were similar in all families studied, the majority of affected family members in the chromosome 5–linked pedigree have a dysarthric speech pattern, which is not present in any of the five unlinked families. The demonstration of heterogeneity within autosomal dominant LGMD is the first step in attempting to subclassify these families with similar clinical phenotypes on a molecular level.     

Identification and functional analysis of a caveolin-3 mutation associated with familial hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are caused by mutations in 14 and 15 different disease genes, respectively, in a part of the patients and the disease genes for cardiomyopathy overlap in part with that for limb-girdle muscular dystrophy (LGMD). In this study, we examined an LGMD gene encoding caveolin-3 (CAV3) for mutation in the patients with HCM or DCM. A Thr63Ser mutation was identified in a sibling case of HCM. Because the mutation was found at the residue that is involved in the LGMD-causing mutations, we investigate the functional change due to the Thr63Ser mutation as compared with the LGMD mutations by examining the distribution of GFP-tagged CAV3 proteins. It was observed that the Thr63Ser mutation reduced the cell surface expression of caveolin-3, albeit the change was mild as compared with the LGMD mutations. These observations suggest that HCM is a clinical spectrum of CAV3 mutations.     

A common haplotype associated with the Basque 2362AG --> TCATCT mutation in the muscular calpain-3 gene

Limb-girdle muscular dystrophy type 2A (LGMD2A) is caused by any of over 150 mutations in the calpain-3 (CAPN3) gene. Of those, 2362AG --> TCATCT is particularly prevalent in Basque patients, and this mutation was hypothesized to have arisen in the Basque Country. To explore the natural history of this mutation, we genotyped 65 Basque and non-Basque patients with LGMD2A who carry the 2362AG --> TCATCT mutation for four microsatellites within or flanking the gene. A particular haplotype was found in three-fourths of the patients and was assumed to be ancestral. From the average number of recombinations and mutations accumulated from this ancestral haplotype, the age of the 2362AG ----> TCATCT mutation was estimated to be 50 generations (i.e., 1,250 years), which is more recent than the Paleolithic Basque heritage. The subsequent spread of the 2362AG --> TCATCT mutation can be related to gene flow out of the Basque Country, even across a cultural border.     

Calpain 3: a key regulator of the sarcomere?

Calpain 3 is a 94-kDa calcium-dependent cysteine protease mainly expressed in skeletal muscle. In this tissue, it localizes at several regions of the sarcomere through binding to the giant protein, titin. Loss-of-function mutations in the calpain 3 gene have been associated with limb-girdle muscular dystrophy type 2A (LGMD2A), a common form of muscular dystrophy found world wide. Recently, significant progress has been made in understanding the mode of regulation and the possible function of calpain 3 in muscle. It is now well accepted that it has an unusual zymogenic activation and that cytoskeletal proteins are one class of its substrates. Through the absence of cleavage of these substrates, calpain 3 deficiency leads to abnormal sarcomeres, impairment of muscle contractile capacity, and death of the muscle fibers. These data indicate a role for calpain 3 as a chef d'orchestre in sarcomere remodeling and suggest a new category of LGMD2 pathological mechanisms.     

Calpainopathy-a survey of mutations and polymorphisms.

Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive disorder characterized mainly by symmetrical and selective atrophy of the proximal limb muscles. It derives from defects in the human CAPN3 gene, which encodes the skeletal muscle-specific member of the calpain family. This report represents a compilation of the mutations and variants identified so far in this gene. To date, 97 distinct pathogenic calpain 3 mutations have been identified (4 nonsense mutations, 32 deletions/insertions, 8 splice-site mutations, and 53 missense mutations), 56 of which have not been described previously, together with 12 polymorphisms and 5 nonclassified variants. The mutations are distributed along the entire length of the CAPN3 gene. Thus far, most mutations identified represent private variants, although particular mutations have been found more frequently. Knowledge of the mutation spectrum occurring in the CAPN3 gene may contribute significantly to structure/function and pathogenesis studies. It may also help in the design of efficient mutation-screening strategies for calpainopathies.     

Mutations in calpain 3 associated with limb girdle muscular dystrophy: analysis by molecular modeling and by mutation in m-calpain

Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive disorder characterized by selective atrophy of the proximal limb muscles. Its occurrence is correlated, in a large number of patients, with defects in the human CAPN3 gene, a gene that encodes the skeletal muscle-specific member of the calpain family, calpain 3 (or p94). Because calpain 3 is difficult to study due to its rapid autolysis, we have developed a molecular model of calpain 3 based on the recently reported crystal structures of m-calpain and on the high-sequence homology between p94 and m-calpain (47% sequence identity). On the basis of this model, it was possible to explain many LGMD2A point mutations in terms of calpain 3 inactivation, supporting the idea that loss of calpain 3 activity is responsible for the disease. The majority of the LGMD2A mutations appear to affect domain/domain interaction, which may be critical in the assembly and the activation of the multi-domain calpain 3. In particular, we suggest that the flexibility of protease domain I in calpain 3 may play a critical role in the functionality of calpain 3. In support of the model, some clinically observed calpain 3 mutations were generated and analyzed in recombinant m-calpain. Mutations of residues forming intramolecular domain contacts caused the expected loss of activity, but mutations of some surface residues had no effect on activity, implying that these residues in calpain 3 may interact in vivo with other target molecules. These results contribute to an understanding of structure-function relationships and of pathogenesis in calpain 3.     

Patients with a non-dysferlin Miyoshi myopathy have a novel membrane repair defect

Two autosomal recessive muscle diseases, limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM), are caused by mutations in the dysferlin gene. These mutations result in poor ability to repair cell membrane damage, which is suggested to be the cause for this disease. However, many patients who share clinical features with MM-type muscular dystrophy do not carry mutations in dysferlin gene. To understand the basis of MM that is not due to mutations in dysferlin gene, we analyzed cells from patients in one such family. In these patients, we found no defects in several potential candidates - annexin A2, caveolin-3, myoferlin and the MMD2 locus on chromosome 10p. Similar to dysferlinopathy, these cells also exhibit membrane repair defects and the severity of the defect correlated with severity of their disease. However, unlike dysferlinopathy, none of the conventional membrane repair pathways are defective in these patient cells. These results add to the existing evidence that cell membrane repair defect may be responsible for MM-type muscular dystrophy and indicate that a previously unsuspected genetic lesion that affects cell membrane repair pathway is responsible for the disease in the non-dysferlin MM patients.     

NP-detecting DNA technologies: solving problems of applied biochemistry

Heterozygosity of CANP3, ACTN3, and GHR genes in specialized collections was studied using state-of-the-art DNA technologies for DNA analysis. A new dinucleotide deletion (AC) at the beginning of exon 21 was identified in five individuals with heterozygous CANP3 gene. Analysis of polymorphism (SNP1747 C-->T) of ACTN3 gene demonstrated a positive association of allele C with a high muscular performance. Real-time PCR assay of SNP1630 (A-->C) in GHR gene suggested a putative negative association of allele C of this SNP with a high muscular performance.     

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

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

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.     

Interactions with M-band Titin and Calpain 3 Link Myospryn (CMYA5) to Tibial and Limb-girdle Muscular Dystrophies

Mutations in the C terminus of titin, situated at the M-band of the striated muscle sarcomere, cause tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy (LGMD) type 2J. Mutations in the protease calpain 3 (CAPN3), in turn, lead to LGMD2A, and secondary CAPN3 deficiency in LGMD2J suggests that the pathomechanisms of the diseases are linked. Yeast two-hybrid screens carried out to elucidate the molecular pathways of TMD/LGMD2J and LGMD2A resulted in the identification of myospryn (CMYA5, cardiomyopathy-associated 5) as a binding partner for both M-band titin and CAPN3. Additional yeast two-hybrid and coimmunoprecipitation studies confirmed both interactions. The interaction of myospryn and M-band titin was supported by localization of endogenous and transfected myospryn at the M-band level. Coexpression studies showed that myospryn is a proteolytic substrate for CAPN3 and suggested that myospryn may protect CAPN3 from autolysis. Myospryn is a muscle-specific protein of the tripartite motif superfamily, reported to function in vesicular trafficking and protein kinase A signaling and implicated in the pathogenesis of Duchenne muscular dystrophy. The novel interactions indicate a role for myospryn in the sarcomeric M-band and may be relevant for the molecular pathomechanisms of TMD/LGMD2J and LGMD2A.     

Oxidative Stress, NF-κB and the Ubiquitin Proteasomal Pathway in the Pathology of Calpainopathy

The neuromuscular disorder, calpainopathy (LGMD 2A), is a major muscular dystrophy classified under limb girdle muscular dystrophies. Genetic mutations of the enzyme calpain 3 cause LGMD 2A. Calpainopathy is phenotypically observed as progressive muscle wasting and weakness. Pathomechanisms of muscle wasting of calpainopathy remain poorly understood. Oxidative stress, NF-κB and the ubiquitin proteasomal pathway underlie the pathology of several muscle wasting conditions but their role in calpainopathic dystrophy is not known. Oxidative and nitrosative stress, the source of reactive oxygen species, NF-κB signaling and protein ubiquitinylation were studied in 15 calpainopathic and 8 healthy control human muscle biopsies. Oxidative stress and NF-κB/IKK β signaling were increased in calpainopathic muscle and may contribute to increased protein ubiquitinylation and muscle protein loss. Preventing oxidative stress or inhibition of NF-κB signaling could be considered for treatment of LGMD 2A.     

Confirmation of genetic heterogeneity in limb-girdle muscular dystrophy: Linkage of an autosomal dominant form to chromosome 5q

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.     

Sarcoglycanopathies: Clinical,Molecular and Genetic Characteristics,Epidemiology, Diagnostics and Treatment Options

Sarcoglycanopathies are a group of autosomal recessive limb-girdle muscular dystrophies (LGMD) caused by mutations in sarcoglycan genes: SGCA (LGMD 2D, MIM 600119), SGCB (LGMD 2E, MIM 604286), SGCG (LGMD 2C, MIM 353700), and SGCD (LGMD 2F, MIM 601287). These genes encode four transmembrane sarcoglycan subunits participating in formation of the large sarcolemmal dystrophin- glycoprotein complex. Clinical symptoms of sarcoglycanopathies resemble the ones in Duchenne/Becker muscular dystrophy and several autosomal recessive LGMD, which causes difficulties in the differential diagnostics between these diseases. This review covers the main aspects of sarcoglycanopathies, such as etiology, spectrum of mutations, clinical features and diagnostics. In addition, we review the fundamental pathogenesis mechanisms leading to sarcoglycanopathies, which can also help to understand the potential options for treatment for patients with muscular dystrophies.     

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.     

Muscle Protein Alterations in LGMD2I Patients With Different Mutations in the Fukutin-related Protein Gene

Fukutin-related protein (FKRP) is a protein involved in the glycosylation of cell surface molecules. Pathogenic mutations in the FKRP gene cause both the more severe congenital muscular dystrophy Type 1C and the milder Limb-Girdle Type 2I form (LGMD2I). Here we report muscle histological alterations and the analysis of 11 muscle proteins: dystrophin, four sarcoglycans, calpain 3, dysferlin, telethonin, collagen VI, α-DG, and α2-laminin, in muscle biopsies from 13 unrelated LGMD2I patients with 10 different FKRP mutations. In all, a typical dystrophic pattern was observed. In eight patients, a high frequency of rimmed vacuoles was also found. A variable degree of α2-laminin deficiency was detected in 12 patients through immunofluorescence analysis, and 10 patients presented α-DG deficiency on sarcolemmal membranes. Additionally, through Western blot analysis, deficiency of calpain 3 and dystrophin bands was found in four and two patients, respectively. All the remaining proteins showed a similar pattern to normal controls. These results suggest that, in our population of LGMD2I patients, different mutations in the FKRP gene are associated with several secondary muscle protein reductions, and the deficiencies of α2-laminin and α-DG on sections are prevalent, independently of mutation type or clinical severity. (J Histochem Cytochem 56:995–1001, 2008)