Autosomal Recessive Limb-girdle Muscular Dystrophy Research Articles

Sarcoglycanopathies: Can muscle immunoanalysis predict the genotype?

Muscle immunoanalysis of the sarcoglycan complex is an important part of the diagnostic evaluation of muscle biopsies in patients with autosomal recessive limb-girdle muscular dystrophy. Reduced or absent sarcolemmal expression of one or all of the four sarcoglycans (α-, β-, γ-, δ-sarcoglycan) can be found in patients with limb-girdle muscular dystrophy 2C-F (LGMD2C-F) and also in patients with Duchenne and Becker muscular dystrophy (DMD/BMD). It has previously been suggested that different patterns of sarcoglycan expression could predict the primary genetic defect, and that genetic analysis could be directed by these patterns. In this first UK study we studied 24 genetically characterized patients with sarcoglycan deficient LGMD, in 22 of whom muscle immunoanalysis data were available. Thirteen patients showed α-sarcoglycan deficient LGMD2D, 7 patients β-sarcoglycan deficient LGMD2E, 3 patients γ-sarcoglycan deficient LGDM2C, and one patient δ-sarcoglycan deficient LGMD2F. Muscle biopsies were analysed in one centre without knowledge of the established genetic diagnosis. Our results demonstrated that residual sarcoglycan expression is highly variable and does not enable an accurate prediction of the genotype. Considering previous reports of sarcoglycanopathy patients with an isolated loss of one sarcoglycan we recommend to use antibodies against all four sarcoglycans for immunoanalysis of skeletal muscle sections. A concomitant reduction of dystrophin and β-dystroglycan was observed more frequently than previously reported and illustrates the important differential diagnosis of DMD and BMD for sarcoglycan deficient LGMD.

Therapeutic Possibilities in the Autosomal Recessive Limb-Girdle Muscular Dystrophies

Fourteen years ago, the first disease-causing mutation in a form of autosomal recessive limb-girdle muscular dystrophy was reported. Since then the number of genes has been extended to at least 14 and the phenotypic spectrum has been broadened. The generation of mouse models helped to improve our understanding of the pathogenesis of the disease and also served to study therapeutic possibilities. All autosomal recessive limb-girdle muscular dystrophies are rare diseases, which is one reason why there have been so very few controlled clinical trials. Other reasons are insufficient natural history data and the lack of standardized assessment criteria and validated outcome measures. Currently, therapeutic possibilities are mainly restricted to symptomatic treatment and the treatment of disease complications. On the other hand, new efforts in translational research and the development of molecular therapeutic approaches suggest that more promising clinical trials will be carried out in autosomal recessive limb-girdle muscular dystrophy in the next several years.

Limb–girdle muscular dystrophies

The aim of this review is to provide an up-to-date analysis of current knowledge about limb-girdle muscular dystrophies (LGMDs). Over the last few years, new and interesting studies have been published on LGMD. New LGMD genes have been discovered and the clinical and genetic heterogeneity in this group of muscular dystrophies has been further enlarged by the description of new forms of LGMD. Several studies have demonstrated involvement of genes causing posttranslational modifications of alpha-dystroglycan in the pathogenesis of autosomal recessive LGMD. This has highlighted an important overlap in pathogenesis between LGMD and congenital muscular dystrophies, prompting further research. Moreover, new pathogenic mechanisms and pathways are emerging for LGMD, in particular calpainopathies, dysferlinopathies and titinopathies. Such new findings may suggest novel therapeutic approaches and future clinical trials. The increased understanding of the genes and pathogenic mechanism of the LGMDs will improve diagnostic processes and prognostic accuracy, and promote therapeutic strategies. European and global LGMD patient registries will increase current knowledge on natural history and facilitate translational research.

Recombinant Adeno-Associated Virus Type 8-Mediated Extensive Therapeutic Gene Delivery into Skeletal Muscle ofα-Sarcoglycan-Deficient Mice

Autosomal recessive limb-girdle muscular dystrophy type 2D (LGMD 2D) is caused by mutations in the alpha-sarcoglycan gene (alpha-SG). The absence of alpha-SG results in the loss of the SG complex at the sarcolemma and compromises the integrity of the sarcolemma. To establish a method for recombinant adeno-associated virus (rAAV)-mediated alpha-SG gene therapy into alpha-SG-deficient muscle, we constructed rAAV serotypes 2 and 8 expressing the human alpha-SG gene under the control of the ubiquitous cytomegalovirus promoter (rAAV2-alpha-SG and rAAV8-alpha-SG). We compared the transduction profiles and evaluated the therapeutic effects of a single intramuscular injection of rAAVs into alpha-SG-deficient (Sgca(-/-)) mice. Four weeks after rAAV2 injection into the tibialis anterior (TA) muscle of 10-day-old Sgca(-/-) mice, transduction of the alpha-SG gene was localized to a limited area of the TA muscle. On the other hand, rAAV8-mediated alpha-SG expression was widely distributed in the hind limb muscle, and persisted for 7 months without inducing cytotoxic and immunological reactions, with a reversal of the muscle pathology and improvement in the contractile force of the Sgca(-/-) muscle. This extensive rAAV8-mediated alpha-SG transduction in LGMD 2D model animals paves the way for future clinical application.

Anesthetic Management of a Woman With Autosomal Recessive Limb-girdle Muscular Dystrophy for Emergency Cesarean Section

Anesthetic Management of a Woman With Autosomal Recessive Limb-girdle Muscular Dystrophy for Emergency Cesarean Section

Revised spectrum of mutations in sarcoglycanopathies

To define the spectrum of mutations in alpha-, beta-, gamma-, and delta-sarcoglycan (SG) genes, we analyzed these genes in 69 probands with clinical and biological criteria compatible with the diagnosis of autosomal recessive limb-girdle muscular dystrophy. For 48 patients, muscle biopsies were available and multiplex western blot analysis of muscle proteins showed significant abnormalities of alpha- and gamma-SG. Our diagnostic strategy includes multiplex western blot, sequencing of SG genes, multiplex quantitative-fluorescent PCR and RT-PCR analyses. Mutations were detected in 57 patients and homozygous or compound heterozygous mutations were identified in 75% (36/48) of the patients with abnormal western blot, and in 52% (11/21) of the patients without muscle biopsy. Involvement of alpha-SG was demonstrated in 55.3% of cases (26/47), whereas gamma- and beta-SG were implicated in 25.5% (12/47) and in 17% (8/47) of cases, respectively. Interestingly, we identified 25 novel mutations, and a significant proportion of these mutations correspond to deletions (identified in 14 patients) of complete exon(s) of alpha- or gamma-SG genes, and partial duplications (identified in 5 patients) of exon 1 of beta-SG gene. This study highlights the high frequency of exonic deletions of alpha- and gamma-SG genes, as well as the presence of a hotspot of duplications affecting exon 1 of the beta-SG gene. In addition, protein analysis by multiplex western blot in combination with mutation screening and genotyping results allowed to propose a comprehensive and efficient diagnostic strategy and strongly suggested the implication of additional genes, yet to be identified, in sarcoglycanopathy-like disorders.

Transcriptional explorations of CAPN3 identify novel splicing mutations, a large‐sized genomic deletion and evidence for messenger RNA decay

Mutations in the gene encoding calpain-3 (CAPN3) cause autosomal recessive limb-girdle muscular dystrophy type 2A (LGMD2A) and idiopathic eosinophilic myositis. Accurate diagnosis and genetic counselling are based on the identification of disease-causing mutations on both alleles of CAPN3 in the patients. In the present study, we used transcriptional analysis as a complementary approach for patients suspected of being affected with LGMD2A, in whom initial denaturing high-performance liquid chromatography genomic mutation screening evidenced no or only one CAPN3 mutation obviously considered as disease causing. This allowed to identify and characterize cDNA deletions. Further genomic analysis allowed to determine the origin of these deletions, either as splicing defects caused by intronic mutations or as an internal multi-exonic deletion. In particular, we report two novel CAPN3 mutations (c.1745 + 4_1745 + 7delAGTG in IVS13 and c.2185-16A>G in IVS20) and a recurrent large-sized genomic deletion including exons 2-8 for which genomic breakpoints have been characterized. In addition, our results indicate nonsense-mediated messenger RNA decay as a mechanism for under-expression of CAPN3 associated to some specific variations.

G.P.4.15 CAPN3 mutations in patients with idiopathic eosinophilic myositis: A predystrophic stage of LGMD2A?

Objective and Background: Mutations in the gene encoding calpain-3 (CAPN3) cause autosomal-recessive limb girdle muscular dystrophy type 2A (LGMD2A). In six children, we recently identified CAPN3 mutations as a genetic cause of idiopathic eosinophilic myositis (EM), a rare pathological entity characterised by eosinophilic infiltration of skeletal muscles. Here, we further evaluated the hypothesis of EM being a possible histopathological pre-dystrophic developmental stage of LGMD2A. Methods: We screened CAPN3 for mutations using DHPLC and direct sequencing, in five unrelated patients (four children and one young adult), recruited on the basis of EM diagnosed after histological examination of muscle biopsy samples, without any identified aetiological factor. In addition, information on the presence of eosinophils was retrospectively assessed in 17 genetically confirmed LGMD2A patients and inflammatory changes by analysing haematoxylin-eosin stained cryosections from muscle samples (mean age of biopsy 28 years). Results: Two disease-causing CAPN3 mutations were identified in the five patients affected with idiopathic EM. In one additional patient, only one disease-causing CAPN3 mutation was identified. Additionally, we found a sparse presence of eosinophils in muscle biopsies from 5/17 patients with inflammatory (n = 3 mild focal, n = 2 moderate focal) and myopathic changes (n = 2 mild, n = 2 moderate, n = 1 severe). Conclusion: Our findings confirm mutations in CAPN3 as a genetic cause of eosinophilic myositis. The presence of eosinophils has been discussed before as a component of muscular dystrophy (Cai et al. 2000). The presence of marked eosinophilic infiltrates in EM patients, together with the findings of our retrospective analysis of biopsies from LGMD2A patients might suggest the following possibilities (1) EM associated with CAPN3 mutations could be a distinct phenotype, (2) eosinophilic infiltration might represent an early or transient developmental stage in the inflammation associated with primary calpainopathy.

Anaesthetic management of a woman with autosomal recessive limb-girdle muscular dystrophy for emergency caesarean section

We report the successful management of a 28-year-old parturient with an autosomal recessive limb-girdle muscular dystrophy with severe restrictive lung disease, who required non-invasive positive pressure ventilation in the third trimester of her pregnancy. At 37 weeks' gestation she had combined spinal-epidural anaesthesia for emergency caesarean section and had an uncomplicated intra- and postoperative course. The anaesthetic management is discussed and the literature briefly reviewed.

Frequency of calpain-3 c.550delA mutation in limb girdle muscular dystrophy type 2 and isolated hyperCKemia in German patients

Calpain-3 deficiency is the most common cause of autosomal-recessive limb girdle muscular dystrophy (LGMD2). The c.550delA mutation in the CAPN3 gene was frequently identified in LGMD2A patients from Eastern Europe and is considered a Slavic founder mutation. We screened for the c.550delA mutation in unrelated German patients with LGMD2 (n = 98) and in patients with asymptomatic or minimally symptomatic (myalgia or fatigue) hyperCKemia of unknown origin (n = 102). Results of Western blot analysis were available in 75 patients with LGMD2 and 65 patients with hyperCKemia. In samples that were heterozygous for the c.550delA mutation, the whole CAPN3 gene was analyzed by sequencing in order to detect the second mutation. The c.550delA mutation was found in 8.1% of LGMD2 (n = 1 homozygous, n = 7 heterozygous) and 1.9% of hyperCKemia patients (n = 2 heterozygous). In 8 of the 9 hetrozygous patients, a second CAPN3 mutation was identified by direct sequencing. Two mutations (Val509Phe and Gln565Stop) have not been reported before. Absent or deficient calpain-3 protein in Western blot analysis was found in 22.5% of the LGMD2 patients and 11% of the patients with hyperCKemia. Western blot results were available in 9 out of the 10 patients with genetically confirmed LGMD2A and were clearly abnormal in 6 patients, suspicious in 2 and entirely normal in 1. Two LGMD2 patients with the c.550delA mutation and onset within the first 2 decades had joint contractures. Muscle biopsy revealed inflammatory changes in three patients. The CAPN3 gene mutation c.550delA is rather frequently observed in German patients with LGMD2, but also occasionally in cases with isolated hyperCKemia.

Structural and functional analysis of the sarcoglycan–sarcospan subcomplex

Sarcospan is a component of the dystrophin–glycoprotein complex that forms a tight subcomplex with the sarcoglycans. The sarcoglycan–sarcospan subcomplex functions to stabilize α-dystroglycan at the plasma membrane and perturbations of this subcomplex are associated with autosomal recessive limb-girdle muscular dystrophy. In order to characterize protein interactions within this subcomplex, we first demonstrate that sarcospan forms homo-oligomers within the membrane. Experiments with a panel of site-directed mutants reveal that proper structure of the large extracellular loop is an important determinant of oligo formation. Furthermore, the intracellular N- and C-termini contribute to stability of sarcospan-mediated webs. Point mutation of each cysteine residue reveals that Cys 162 and Cys 164 within the large extracellular loop form disulfide bridges, which are critical for proper sarcospan structure. The extracellular domain of sarcospan also forms the main binding site for the sarcoglycans. We propose a model whereby sarcospan forms homo-oligomers that cluster the components of the dystrophin–glycoprotein complex within the membrane.

A novel autosomal recessive limb-girdle muscular dystrophy with quadriceps atrophy maps to 11p13-p12

Limb-girdle muscular dystrophies (LGMD) are a heterogeneous group of pathologies. We have identified a cohort of 14 French-Canadian patients from eight different families displaying a novel form of LGMD with an autosomal recessive inheritance. These patients share some features with previously described cases of 'quadriceps myopathy' that evolved into an LGMD. All demonstrate quadriceps femoris asymmetrical atrophy. Creatine kinase values were variable from normal to 6000 U/l. Clinical evaluations and MRI studies demonstrate a variable intrafamilial and interfamilial phenotype. Asymmetrical muscle involvement was clinically observed and confirmed by imaging. MRI studies suggest that the hamstrings and the adductor magnus are the first limb muscles to demonstrate fatty infiltration. Muscle pathology shows no sign of active inflammation but increased endomysial connective tissue associated with basal lamina duplication and collagen disorganization. A genome-wide scan using the two largest families uncovered linkage to marker D11S1360 on chromosome 11p12 [multipoint logarithm of the odds (LOD) score of 2.78]. Further genotyping for the eight families confirmed linkage to this new LGMD locus (multipoint LOD score of 4.56). Fine mapping subsequently defined a less than 3.3 cM candidate interval on 11p13-p12. Haplotype analysis of carrier chromosomes suggests that the most frequent mutation may account for up to 81.3% of French-Canadian mutations. In this study, we describe the chromosomal locus of a new form of recessive LGMD with prominent quadriceps femoris atrophy.

P.O.4 A novel autosomal recessive limb-girdle muscular dystrophy with quadriceps atrophy maps to 11p13-p12

P.O.4 A novel autosomal recessive limb-girdle muscular dystrophy with quadriceps atrophy maps to 11p13-p12

Cardiac involvement in limb-girdle muscular dystrophy 2I

The C826A mutation in the fukutin-related protein (FKRP) gene is typically associated with autosomal recessive limb-girdle muscular dystrophy 2I (LGMD2I) but oligosymptomatic phenotypes and patients with predominant cardiac involvement are also described. To assess cardiac involvement in patients with LGMD2I. Nine patients from 5 families (2 female, 7 male) homozygous for the 826C > A FKRP mutation were included. Additional to conventional cardiac investigations (electrocardiography and echocardiography) the patients underwent cardiovascular magnetic resonance imaging (CMR). Cardiac involvement was detected by CMR in eight of nine patients (reduced left ventricular ejection fraction in 6, enlargement of left ventricular end-diastolic volume in 2 and left ventricular mass in 2) and in four patients by conventional cardiac diagnostic investigations. Two of the nine patients showed no muscle weakness or atrophy but suffered myalgias; both had cardiac manifestation of the disease. CMR is a sensitive method for detecting cardiac abnormalities in patients with LGMD2I and can be used for early detection of mild or subclinical cardiac involvement.

Identification of functional domains in sarcoglycans essential for their interaction and plasma membrane targeting

Mutations in sarcoglycans have been reported to cause autosomal-recessive limb-girdle muscular dystrophies. In skeletal and cardiac muscle, sarcoglycans are assembled into a complex on the sarcolemma from four subunits (α, β, γ, δ). In this report, we present a detailed structural analysis of sarcoglycans using deletion study, limited proteolysis and co-immunoprecipitation. Our results indicate that the extracellular regions of sarcoglycans consist of distinctive functional domains connected by proteinase K-sensitive sites. The N-terminal half domains are required for sarcoglycan interaction. The C-terminal half domains of β-, γ- and δ-sarcoglycan consist of a cysteine-rich motif and a previously unrecognized conserved sequence, both of which are essential for plasma membrane localization. Using a heterologous expression system, we demonstrate that missense sarcoglycan mutations affect sarcoglycan complex assembly and/or localization to the cell surface. Our data suggest that the formation of a stable complex is necessary but not sufficient for plasma membrane targeting. Finally, we provide evidence that the β/δ-sarcoglycan core can associate with the C-terminus of dystrophin. Our results therefore generate important information on the structure of the sarcoglycan complex and the molecular mechanisms underlying the effects of various sarcoglycan mutations in muscular dystrophies.

Calpain-3 mutations in Turkey

Autosomal recessive limb-girdle muscular dystrophies (LGMD2s) are a clinically and genetically heterogeneous group of disorders, characterized by progressive involvement of the proximal limb girdle muscles; the group includes at least 10 different genetic entities. The calpainopathies (LGMD2A), a subgroup of LGMD2s, are estimated to be the most common forms of LGMD2 in all populations so far investigated. LGMD2A is usually characterized by symmetrical and selective atrophy of pelvic, scapular and trunk muscles and a moderate to gross elevation of serum CK. However, the course is highly variable. It is caused by mutations in the CAPN3 gene, which encodes for the calpain-3 protein. Until now, 161 pathogenic mutations have been found in the CAPN3 gene. In the present study, through screening of 93 unrelated LGMD2 families, we identified 29 families with LGMD2A, 21 (22.6%) of which were identified as having CAPN3 gene mutations. We detected six novel (p.K211N, p.D230G, p.Y322H, p.R698S, p.Q738X, c.2257delGinsAA) and nine previously reported mutations (c.550delA, c.19_23del, c.1746-20C>G, p.R49H, p.R490Q, p.Y336N, p.A702V, p.Y537X, p.R541Q) in the CAPN3 gene. There may be a wide variety of mutations, but clustering of specific mutations (c.550delA: 40%, p.R490Q: 10%) could be used in the diagnostic scheme in Turkey.

Frequency of calpain-3 c.550delA mutation in limb girdle muscular dystrophy type 2 and isolated hyperCKemia in German patients

Aims: Calpain-3 deficiency is the most common cause of autosomal-recessive limb-girdle muscular dystrophy (LGMD2). The 550delA mutation in the CAPN3 gene was frequently identified in patients from Eastern Europe.

Limb girdle muscular dystrophy type 2I caused by a novel missense mutation in the FKRP gene presenting as acute virus-associated myositis in infancy

Limb girdle muscular dystrophies (LGMD) are a clinically and genetically heterogenous group of disorders characterised by weakness and wasting of predominantly the pelvic and shoulder girdle muscles [2,3]. Mutations in the recently identified fukutin-related protein gene FKRP (MIM 606596), which encodes a putative glycosyltransferase, cause LGMD type 2I (LGMD21) as well as congenital muscular dystrophy (MDC) type 1C [1]. In patients with LGMD2I, apparently the most common form of autosomal-recessive LGMD in Europe, considerable variability of age of onset, progression and cardiorespiratory involvement has been reported [4, 5,6]. We describe the clinical course in two boys (patient 1; patient 2) of healthy, non-consanguineous parents with normal psychomotor development (patient 1 walked at the age of 11 months and patient 2 at the age of 10 months). At the age of 3 (patient 1) and 1.5 (patient 2) years, respectively, they presented with a febrile viral respiratory infection, increasing weakness and an inability to walk. At that time, serum creatin kinase (CK) levels were highly elevated to 111 lmol/l (patient 1) and 328 lmol/l (patient 2) (normal <3 lmol/l). Symptoms resolved after 1 week and CK levels declined within weeks to 10 lmol/l. Clinically, a virus-associated myositis was suspected. During the further course, patient 1 showed a broadbased waddling gait, marked pseudohypertrophy of both calves (Fig. 1a) and marked weakness of the limb girdle and thighs. His CK level was elevated to 72 lmol/l at the age of 8 years and the histopathological examination of a quadriceps muscle biopsy specimen revealed histological changes characteristic of a muscular dystrophy with normal expression of dystrophin (antibodies Dys1-Dys3). At the age of 14 years, he presented with a dilated cardiomyopathy (fractional shortening of the left ventricle of 16%, enddiastolic left ventricular diameter of 60 mm) and was commenced on an angiotensin converting enzyme inhibitor. His brother exhibited mild proximal leg weakness and pseudohypertrophy of both calves (Fig. 1b), but no restrictions in walking distance

-Sarcoglycan compensates for lack of -sarcoglycan in a mouse model of limb-girdle muscular dystrophy

Dystrophin and the dystrophin-associated protein (DAP) complex protect the sarcolemma against contraction-induced injury and serve as a mechanical link between the extracellular matrix and the actin cytoskeleton. Some of the functional properties of the DAP complex are mediated by its sarcoglycan (SG) subcomplex, which is composed of alpha-, beta-, gamma- and delta-SGs. Autosomal recessive limb-girdle muscular dystrophy type-2D (LGMD 2D) results from reduction in SG subcomplex levels caused by specific mutations in the muscle-specific alpha-SG gene. epsilon-SG is a widely expressed homolog of the muscle-specific alpha-SG, and expression of epsilon-SG may compensate for the pathologic changes in alpha-SG function. Thus, the goal of the present study was to investigate whether overexpression of epsilon-SG can compensate for dysfunction of alpha-SG. Several transgenic mouse lines that overexpress epsilon-SG in skeletal muscle were established. Overexpression of epsilon-SG in normal mice resulted in substitution of epsilon-SG for alpha-SG in the SG complex of skeletal muscle without any obvious abnormalities. To determine whether an increase in epsilon-SG expression may prevent muscular dystrophy in the context of alpha-SG-deficiency, these epsilon-SG transgenic mice were crossed with alpha-SG deficient mice. alpha-SG-deficient mice overexpressing epsilon-SG exhibited no skeletal muscle cell membrane damage or abnormal contraction. These data suggest that the overexpression of epsilon-SG may represent a therapeutic strategy for treatment of LGMD 2D.

Specific assembly pathway of sarcoglycans is dependent on beta- and delta-sarcoglycan.

Mutations in sarcoglycans (SG) have been reported to cause autosomal-recessive limb-girdle muscular dystrophy (LGMD) and dilated cardiomyopathy. In skeletal and cardiac muscle, sarcoglycans exist as a complex of four transmembrane proteins (alpha-, beta-, gamma-, and delta-SG). In this study, the assembly of the sarcoglycan complex was examined in a heterologous expression system. Our results demonstrated that the assembly process occurs as a discrete stepwise process. We found that beta-SG appears to play an initiating role and its association with delta-SG is essential for the proper localization of the sarcoglycan complex to the cell membrane. The incorporation of alpha-SG into the sarcoglycan complex occurs at the final stage by interaction with gamma-SG. These findings were supported by chemical cross-linking of endogenous sarcoglycans in cultured myotubes. We have also provided evidence that glycosylation-defective mutations in beta-SG and a common mutation in gamma-SG (C283Y) disrupt sarcoglycan-complex formation. Our proposed model for the assembly and structure of sarcoglycans should generate important insight into their function in muscle as well as their role in muscular dystrophies and cardiomyopathies.