Clinical correlations and long-term follow-up in 100 patients with sarcoglycanopathies.

BACKGROUND AND PURPOSE: To describe a large series of patients with α, ß, and γ sarcoglycanopathies (LGMD-R3, R4, and R5) and study phenotypic correlations and disease progression. METHODS: A multicentric retrospective study in four centers in the Paris area collecting neuromuscular, respiratory, cardiac, histologic, and genetic data. The primary outcome of progression was age of loss of ambulation (LoA); disease severity was established according to LoA before or after 18 years of age. Time-to-event analysis was performed. RESULTS: One hundred patients (54 γ-SG; 41 α-SG; 5 ß-SG) from 80 families were included. The γ-SG patients had earlier disease onset than α-SG patients (5.5 vs. 8 years; p = 0.022) and ß-SG patients (24.4 years). Axial muscle weakness and joint contractures were frequent and exercise intolerance was observed. At mean follow-up of 22.9 years, 65.3% of patients were wheelchair-bound (66.7% α-SG, 67.3% γ-SG, 40% ß-SG). Dilated cardiomyopathy occurred in all sarcoglycanopathy subtypes, especially in γ-SG patients (p = 0.01). Thirty patients were ventilated and six died. Absent sarcoglycan protein expression on muscle biopsy and younger age at onset were associated with earlier time to LoA (p = 0.021 and p = 0.002). Age at onset was an independent predictor of both severity and time to LoA (p = 0.0004 and p = 0.009). The α-SG patients showed genetic heterogeneity, whereas >90% of γ-SG patients carried the homozygous c.525delT frameshift variant. Five new mutations were identified. CONCLUSIONS: This large multicentric series delineates the clinical spectrum of patients with sarcoglycanopathies. Age at disease onset is an independent predictor of severity of disease and LoA, and should be taken into account in future clinical trials.

Novel CAPN3 variant associated with an autosomal dominant calpainopathy.

AIMS: The most common autosomal recessive limb girdle muscular dystrophy is associated with the CAPN3 gene. The exclusively recessive inheritance of this disorder has been recently challenged by the description of the recurrent variants, c.643_663del21 [p.(Ser215_Gly221del)] and c.598_612del15 [p.(Phe200_Leu204del)], associated with autosomal dominant inheritance. Our objective was to confirm the existence of autosomal dominant calpainopathies. METHODS: Through our activity as one of the reference centres for genetic diagnosis of calpainopathies in France and the resulting collaborations through the French National Network for Rare Neuromuscular Diseases (FILNEMUS), we identified four families harbouring the same CAPN3 heterozygous variant with supposedly autosomal dominant inheritance. RESULTS: We identified a novel dominantly inherited CAPN3 variant, c.1333G>A [p.(Gly445Arg)] in 14 affected patients from four unrelated families. The complementary phenotypic, functional and genetic findings correlate with an autosomal dominant inheritance in these families, emphasizing the existence of this novel transmission mode for calpainopathies. The mild phenotype associated with these autosomal dominant cases widens the phenotypic spectrum of calpainopathies and should therefore be considered in clinical practice. CONCLUSIONS: We confirm the existence of autosomal dominant calpainopathies as an entity beyond the cases related to the in-frame deletions c.643_663del21 and c.598_612del15, with the identification of a novel dominantly inherited and well-documented CAPN3 missense variant, c.1333G>A [p.(Gly445Arg)]. In addition to the consequences for genetic counselling, the confirmation of an autosomal dominant transmission mode for calpainopathies underlines the importance of re-assessing other myopathies for which the inheritance is considered as strictly autosomal recessive.

Diagnostic anoctamin-5 protein defect in patients with ANO5-mutated muscular dystrophy.

AIMS: Previously, detection of ANO5 protein has been complicated by unspecific antibodies, most of which have not identified the correct protein. The aims of the study were to specify ANO5 protein expression in human skeletal muscle, and to investigate if the ANO5 protein levels are affected by different ANO5 mutations in anoctaminopathy patients. METHODS: Four different antibodies were tested for ANO5 specificity. A sample preparation method compatible with membrane proteins, combined with tissue fractionation was used to determine ANO5 expression in cell cultures expressing ANO5, in normal muscles and eight patient biopsies with six different ANO5 mutations in homozygous or compound heterozygous states, and in other dystrophies. RESULTS: Only one specific monoclonal N-terminal ANO5 antibody was efficient in detecting the protein, showing that ANO5 is expressed as a single 107 kD polypeptide in human skeletal muscle. The truncating mutations c.191dupA and c.1261C>T were found to abolish ANO5 expression, whereas the studied point mutations had variable effects; however, all the ANO5 mutations resulted in clearly reduced ANO5 expression in the patient muscle membrane fraction. Attempts to detect ANO5 using immunohistochemistry were not yet successful. CONCLUSIONS: The data presented here indicate that the ANO5 protein expression is decreased in ANO5-mutated muscular dystrophy and that most of the non-truncating pathogenic ANO5 mutations likely destabilize the protein and cause its degradation. The method described here allows direct analysis of human ANO5 protein, which can be used in diagnostics, for evaluating the pathogenicity of the potentially harmful ANO5 variants of uncertain significance.

Muscle involvement in limb-girdle muscular dystrophy with GMPPB deficiency (LGMD2T).

OBJECTIVE: In this study, muscle involvement assessed by MRI and levels of GMPPB and glycosylation of α-dystroglycan expression in muscle were examined in patients with limb-girdle muscular dystrophy (LGMD) type 2T. METHODS: Six new patients with genetically verified mutations in GMPPB were studied. T1-weighted magnetic resonance images were obtained in 4 participants. Muscle strength and potential involvement of extramuscular organs were examined. Glycosylation of α-dystroglycan in muscle was studied, and GMPPB and α-dystroglycan expression was analyzed by Western blotting. Prevalence of LGMD2T was calculated from the total LGMD population in Denmark. GMPPB was sequenced in all unclassified cases. RESULTS: Two patients carried 3 new mutations in GMPPB. The other 4 patients carried previously described pathogenic mutations in GMPPB. MRI showed that the paraspinal muscles were the most affected, followed by involvement of hamstrings. Our results showed a loss of glycosylation of α-dystroglycan as well as secondary loss of merosin expression on Western blotting. The prevalence of LGMD2T in the Danish cohort of patients with LGMD is 1.5%. CONCLUSIONS: The new findings of this study are (1) the consistent finding of a preferential affection of paraspinal and hamstring muscles in LGMD2T, (2) 3 new mutations in GMPPB, (3) variable loss of glycosylation tested with IIH6 and VIA4 antibodies, and (4) a prevalence of LGMD2T of 1.5% in a well-characterized Danish LGMD cohort.

[Genetics and molecular aspects of dystrophinopathies]. / Aspects génétiques et moléculaires des dystrophinopathies.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by mutations in the DMD gene that encodes the cytoskeletal protein, dystrophin. Dystrophinopathies are inherited in an X-linked recessive manner. Due to the tremendous size of the gene (2.2 megabases), the DMD locus has a high spontaneous mutation rate, and one third of sporadic cases of DMD are due to a de novo mutation. There are seven tissue-specific promoters in the gene. The skeletal muscular transcript contains 79 exons and encode the full-length protein (427-kDa) located at the inner face of the sarcolemma of muscle fibers. DMD gene mutations are highly heterogeneous. Large rearrangements (deletions or duplications of one or more exons) are most frequently involved while point mutations account for 20 %-30 % of cases. A survey of current strategies of molecular diagnosis is presented here. In particular, the role of muscle biopsy (for dystrophin and RNA analyses) in the diagnosis of dystrophinopathies is discussed. In more than 90 % of cases, the clinical severity is correlated with the impact of the mutations on the reading frame and the expression of the dystrophin (absence or residual amount of mutated protein). Various mechanisms contribute to the exceptions. Besides the clinical interest for the patient, the identification of the mutation allows accurate genetic counseling in the familles, and is a necessary prerequisite for the inclusion of the patient in the genotype-based clinical trials.

Dilated cardiomyopathy in patients with mutations in anoctamin 5.

BACKGROUND: Homozygous mutations in ANO5, a gene encoding anoctamin 5, a putative calcium-activated chloride channel, have recently been reported in patients with adult-onset myopathies or isolated high-CK levels. Cardiomyopathy has not previously been reported in these populations despite a proven expression of anoctamin 5 in the cardiac muscle. METHODS: Patients presenting for the management of high-CK levels or overt myopathy with proven ANO5 mutations were prospectively investigated between June 2010 and March 2012 in Pitié Salpêtrière Hospital, according to a standardised protocol. Neurological and cardiological clinical examinations, CK assessment, electrocardiogram (ECG), and echocardiography were performed, as well as cardiac MRI and coronary CT angiography in patients with left ventricular (LV) dysfunction. RESULTS: Our study included 19 consecutive patients (male=15, age=46.2 ± 12.7 years) from 16 families. Five had asymptomatic high-CK levels and 14 had overt myopathy. One patient had a personal history of stable coronary artery disease with normal ventricular function. ECG showed ventricular premature beats in one patient. Echocardiography displayed LV dilatation in two patients, LV dysfunction in one, and both abnormalities in two who fulfilled criteria for dilated cardiomyopathy which was confirmed by cardiac MRI and normal CT angiography. CONCLUSIONS: Dilated cardiomyopathy is a potential complication in patients with myopathies due to mutations in the ANO5 gene whose screening requires specific procedures.

Miyoshi-like distal myopathy with mutations in anoctamin 5 gene.

Miyoshi myopathy is the most common form of recessive distal myopathy. Recessive mutations in the ANO5 gene have been recently identified in Northern Europe as a cause of non dysferlin-linked distal myopathy and limb girdle muscular dystrophy. We report here the first French cases of anoctamin 5 myopathy in 2 brothers presenting with a Miyoshi-like pattern. Comparing these patients with 12 other cases from the literature shows that all cases share a homogeneous clinical pattern, characterized by initial calf muscles involvement. Asymmetric muscle atrophy often precedes weakness. In this setting, high CK level and normal expression of dysferlin in muscle should lead to consider the diagnosis, which will be confirmed by ANO5 gene testing. The c.191dupA mutation, already reported as a founder mutation in Caucasian patients with anoctamin myopathies, was found in our family in a heterozygous state.

Immunolabelling and flow cytometry as new tools to explore dysferlinopathies.

Dysferlinopathies are autosomal recessive muscular dystrophies caused by DYSF mutations, which lead to a reduced amount or a complete lack of dysferlin. One step in dysferlinopathies diagnosis consists in Western blot analysis of proteins extracted from muscle biopsy, or blood monocytes. We have taken advantage of dysferlin expression in monocytes to develop a whole blood flow cytometry (WBFC), using antibodies directed against dysferlin. Six patients were submitted to WBFC analysis and immunofluorescence analysis on monocytes. Results obtained are correlated to Western blot from monocytes and muscle biopsies. The possible usefulness of this flow cytometry analysis in routine diagnosis is presented.

Complete fatty degeneration of lumbar erector spinae muscles caused by a primary dysferlinopathy.

Magnetic resonance imaging of the thoracolumbar spine showed complete fatty degeneration of the lumbar erector spinae muscles in a woman who had complained of chronic lower back pain for 5 years and of progressive weakness of the lower limbs for 1 year. Neuromuscular examination of the lower limbs showed no obvious anomaly, and there was no camptocormia. Serum creatine kinase levels were increased (six- to ninefold); electrodiagnostic examination revealed no activity at rest or during effort in the erector spinae muscles and was normal in proximal and distal muscles of the limbs. Muscle computed tomography revealed mild fatty degeneration of thigh and gastrocnemius muscles, and histopathology of the deltoid muscle showed dystrophic features and complete lack of dysferlin. Molecular analysis identified a homozygous disease-causing mutation in the gene encoding dysferlin. Because there were no similar cases in the family, the final diagnosis was sporadic limb-girdle muscular dystrophy type 2B. Overall, this case report shows that the lumbar and lower thoracic of erector spinae muscles may display complete fatty degeneration without the occurrence of camptocormia, with primary dysferlin deficiency as a possible cause.

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.

New POMT2 mutations causing congenital muscular dystrophy: identification of a founder mutation.

BACKGROUND: Dystroglycanopathies are a group of congenital muscular dystrophies (CMDs) with autosomal recessive inheritance, often associated with CNS and ocular involvement. They are characterized by the abnormal glycosylation of alpha-dystroglycan, and caused by mutations in at least six genes encoding enzymes: FKTN, POMGNT1, POMT1, POMT2, FKRP, and LARGE. POMT2 mutations have recently been identified in Walker-Warburg syndrome and in a milder muscle-eye-brain disease-like form. METHODS: We studied mentally retarded patients with CMD, analyzed POMT2 by sequencing the coding regions, and also performed a haplotype analysis in all patients and their family members carrying the new POMT2 mutation. RESULTS: We report three novel POMT2 mutations. One of these, p.Tyr666Cys, was homozygous in two unrelated patients and in a compound heterozygous state in others. All patients showed severe diffuse muscle weakness, microcephaly, severe mental retardation, and marked lordoscoliosis with hyperextended head. Elevated CK levels, cerebral cortical atrophy, and cerebellar vermis hypoplasia were constant findings. Mild cardiac abnormalities, focal white matter abnormalities, or partial corpus callosum hypoplasia were detected in single cases. Eye involvement was absent or mild. By genotype analysis, we defined a distinct 170kb haplotype encompassing POMT2 and shared by all the subjects harboring the mutation p.Tyr666Cys. CONCLUSIONS: Our results broaden the clinical spectrum associated with POMT2 mutations, which should be considered in patients with CMD associated with microcephaly, and severe mental retardation with or without ocular involvement.

[Early detection of myocardial disease in young patients with Becker's muscular dystrophy asymptomatic from the cardiac point of view: value of myocardial doppler tissue imaging]. / Detection précoce de l'atteinte myocardique chez des jeunes patients atteints de dystrophie musculaire de Becker asymptomatique sur le plan cardiaque Intérêt du doppler tissulaire myocardique.

Becker's muscular dystrophy is an X-linked hereditary disorder characterised by progressive muscle weakness and possible cardiac disease. Cardiac involvement is assumed to be rare in young patients. Early diagnosis could lead to earlier treatment at an infra-clinical stage of the disease. The object of the study was to evaluate systolic and diastolic cardiac function of young patients with Becker's disease by echocardiography and using Doppler tissue imaging. Consecutive patients under 20 years of age with Becker's disease confirmed genetically were included and compared with paired normal subjects. Subendocardial and subepicardial myocardial velocities were obtained by Doppler tissue imaging and the corresponding velocity gradients were measured. Twelve patients were included (17.4 +/- 2.5 years). None of them had disabling muscle disease. No significant difference was observed from normal subjects with respect to: ventricular dimensions, wall thickness, fractional shortening, E/A ratio measured by transmitral Doppler. Nevertheless, patients with Becker's disease had lower systolic and diastolic intra-myocardial velocity gradients: 2.2 +/- 1.1 vs. 4.7 +/- 2.4 s(-1), p = 0.006, and 3.6 +/- 2.0 vs. 5.6 +/- 1.3 s(-1), p = 0.048, respectively, compared with the control group. These results show that myocardial disease is possible in patients with Becker's muscular dystrophy under the age of 20. Myocardial Doppler tissue imaging is a sensitive method for detecting these early abnormalities and should be recommended in the young patients.

Multitissular involvement in a family with LMNA and EMD mutations: Role of digenic mechanism?

BACKGROUND: Mutations in the EMD and LMNA genes, encoding emerin and lamins A and C, are responsible for the X-linked and autosomal dominant and recessive forms of Emery-Dreifuss muscular dystrophy (EDMD). LMNA mutations can also lead to several other disorders, collectively termed laminopathies, involving heart, fat, nerve, bone, and skin tissues, and some premature ageing syndromes. METHODS: Fourteen members of a single family underwent neurologic, electromyographic, and cardiologic assessment. Gene mutation and protein expression analyses were performed for lamins A/C and emerin. RESULTS: Clinical investigations showed various phenotypes, including isolated cardiac disease (seven patients), axonal neuropathy (one patient), and a combination of EDMD with axonal neuropathy (two patients), whereas five subjects remained asymptomatic. Genetic analyses identified the coincidence of a previously described homozygous LMNA mutation (c.892C-->T, p. R298C) and a new in-frame EMD deletion (c.110-112delAGA, p. delK37), which segregate independently. Analyses of the contribution of these mutations showed 1) the EMD codon deletion acts in X-linked dominant fashion and was sufficient to induce the cardiac disease, 2) the combination of both the hemizygous EMD and the homozygous LMNA mutations was necessary to induce the EDMD phenotype, 3) emerin was present in reduced amount in EMD-mutated cells, and 4) lamin A/C and emerin expression was most dramatically affected in the doubly mutated fibroblasts. CONCLUSIONS: This highlights the crucial role of lamin A/C-emerin interactions, with evidence for synergistic effects of these mutations that lead to Emery-Dreifuss muscular dystrophy as the worsened result of digenic mechanism in this family.

LGMD2A: genotype-phenotype correlations based on a large mutational survey on the calpain 3 gene.

We present here the clinical, molecular and biochemical findings from 238 limb-girdle muscular dystrophy type 2A (LGMD2A) patients, representing approximately 50% (238 out of 484) of the suspected calpainopathy cases referred for the molecular study of the calpain 3 (CAPN3) gene. The mean age at onset of LGMD2A patients was approximately 14 years, and the first symptoms occurred between 6 and 18 years of age in 71% of patients. The mean age at which the patients became wheelchair bound was 32.2 years, with 84% requiring the use of a wheelchair between the age of 21 and 40 years. There was no correlation between the age at onset and the time at which the patient became wheelchair bound, nor between the sex of the patient and the risk of becoming wheelchair bound. Of the cases where the CAPN3 gene was not affected, approximately 20% were diagnosed as LGMD2I muscular dystrophy, while facioscapulohumeral muscular dystrophy (FSHD) was uncommon in this sample. We identified 105 different mutations in the CAPN3 gene of which 50 have not been described previously. These were distributed throughout the coding region of the gene, although some exons remained free of mutations. The most frequent mutation was 2362AG-->TCATCT (exon 22), which was present in 30.7% of the chromosomes analysed (146 chromosomes). Other recurrent mutations described were N50S, 550DeltaA, G222R, IVS6-1G-->A, A483D, IVS17+1G-->T, 2069-2070DeltaAC, R748Q and R748X, each of which was found in >5 chromosomes. The type of mutation in the CAPN3 gene does not appear to be a risk factor for becoming dependent on a wheelchair at a determined age. However, in the cases with two null mutations, there were significantly fewer patients that were able to walk than in the group of patients with at least one missense mutation. Despite the fact that the results of phenotyping and western blot might be biased due to multiple referral centres, producing a diagnosis on the basis of the classical phenotype is neither sufficiently sensitive (86.7%) nor specific (69.3%), although western blot proved to be even less sensitive (52.5%) yet more specific (87.8%). In this case LGMD2I was a relevant cause of false-positive diagnoses. Considering both the clinical phenotype and the biochemical information together, the probability of correctly diagnosing a calpainopathy is very high (90.8%). However, if one of the analyses is lacking, the probability varies from 78.3 to 73.7% depending on the information available. When both tests are negative, the probability that the sample comes from a patient with LGMD2A was 12.2%.

Beta-sarcoglycanopathy (LGMD 2E) in a Spanish family.

Out of 10 autosomal recessive limb-girdle muscular dystrophies reported, 4 are caused by mutations in the genes encoding for sarcoglycans (alpha-, beta-, gamma- and delta-SG). Beta-sarcoglycanopathy (limb-girdle muscular dystrophy 2E) is a genetically heterogeneous disorder which usually presents a severe progressive clinical course. A complete immunohistochemical evaluation of the sarcoglycan complex should be carried out to direct the mutation analysis approach. The present report concerns a Spanish family with a genetically confirmed beta-sarcoglycanopathy. The patient, a 16-year-old female, offspring of a consanguineous marriage, developed a severe limb-girdle muscular dystrophy with a Duchenne-like phenotype. Muscle biopsy showed dystrophic changes and complete absence of the four sarcoglycans. Genetic analysis demonstrated homozygosis for the M100K missense mutation in exon 3, encoding for the proximal extracellular domain. The parents and one sister were found to be carriers. Missense mutations affecting this domain result in the instability of the entire sarcoglycan complex and lead to severe phenotypes as seen in non-sense mutations.

[Muscular exercise intolerance syndrome in Becker muscular dystrophy]. / Place du syndrome d'intolérance musculaire à l'exercice dans la dystrophie musculaire de Becker.

OBJECTIVE: To determine whether exertional muscle pain syndrome (EMP) is a benign phenotype or an early stage of Becker-type muscular dystrophy (BMD). METHODS: Muscle dystrophin analysis led to the diagnosis of BMD in 6 patients complaining of EMP. RESULTS: Three patients had a history of X-linked inheritance and age at clinical onset was 4 to 11 years in five, and one patient had a later onset aged 23. Pseudohypertrophy of the calf muscles was absent in one patient, but all had experienced mild (5/6) pelvic weakness and (or) atrophy one to 17 years after the onset. High serum CK level was present (X 14). Normal anti-dystrophin immunostaining in two cases did not rule out the diagnosis that was only made made by Western blot analysis or genetic studies. All exhibited in-frame deletions (exons 45-48) within the dystrophin gene. CONCLUSION: The 36 patients with BMD-EMP analysed in the literature, exhibited different deletions and no worsening in 66.7% of cases. Western blot was more precise than immunolabelling with 96.8% positivity versus 70.5%. Dystrophin analysis by Western blot and (or) DNA analysis should be included in the evaluation of patients with EMP syndrome without deficient muscle energy metabolism, particularly those with pseudohypertrophy of the calf muscles or high serum CK levels.

Pseudo-metabolic presentation in a Duchenne muscular dystrophy symptomatic carrier with 'de novo' duplication of dystrophin gene.

We report a 6-year-old female patient presenting with a sudden and severe single episode of rhabdomyolysis in which screening for a metabolic disorder was negative. Four months after the episode a muscle biopsy was performed and showed a mild pattern of necrosis/regeneration. Upon immunofluorescence, a mosaic pattern of dystrophin deficiency was found, and in the dystrophin deficient muscle fibres, the four proteins of the sarcoglycan complex were also lacking. Genetic analysis showed a duplication of exons 3 to 17 on one X-chromosome of the proband, but not on the mother's X-chromosome. A clearly skewed X-inactivation (85% of the defective X being active) was found and is consistent with the patient being symptomatic. To our knowledge, a spontaneous rhabdomyolysis in a female Duchenne muscular dystrophy carrier has never been reported.