miR-379 links glucocorticoid treatment with mitochondrial response in Duchenne muscular dystrophy.

Duchenne Muscular Dystrophy (DMD) is a lethal muscle disorder, caused by mutations in the DMD gene and affects approximately 1:5000-6000 male births. In this report, we identified dysregulation of members of the Dlk1-Dio3 miRNA cluster in muscle biopsies of the GRMD dog model. Of these, we selected miR-379 for a detailed investigation because its expression is high in the muscle, and is known to be responsive to glucocorticoid, a class of anti-inflammatory drugs commonly used in DMD patients. Bioinformatics analysis predicts that miR-379 targets EIF4G2, a translational factor, which is involved in the control of mitochondrial metabolic maturation. We confirmed in myoblasts that EIF4G2 is a direct target of miR-379, and identified the DAPIT mitochondrial protein as a translational target of EIF4G2. Knocking down DAPIT in skeletal myotubes resulted in reduced ATP synthesis and myogenic differentiation. We also demonstrated that this pathway is GC-responsive since treating mice with dexamethasone resulted in reduced muscle expression of miR-379 and increased expression of EIF4G2 and DAPIT. Furthermore, miR-379 seric level, which is also elevated in the plasma of DMD patients in comparison with age-matched controls, is reduced by GC treatment. Thus, this newly identified pathway may link GC treatment to a mitochondrial response in DMD.

Respiratory and upper limb function as outcome measures in ambulant and non-ambulant subjects with Duchenne muscular dystrophy: A prospective multicentre study.

The field of translational research in Duchenne muscular dystrophy (DMD) has been transformed in the last decade by a number of therapeutic targets, mostly studied in ambulant patients. A paucity of studies focus on measures that capture the non-ambulant stage of the disease, and the transition between the ambulant and non-ambulant phase. In this prospective natural history study, we report the results of a comprehensive assessment of respiratory, upper limb function and upper limb muscle strength in a group of 89 DMD boys followed in 3 European countries, 81 receiving corticosteroids, spanning a wide age range (5-18 years) and functional abilities, from ambulant (n = 60) to non-ambulant (n = 29). Respiratory decline could be detected in the early ambulatory phase using Peak Expiratory Flow percentage predicted (PEF%), despite glucocorticoid use (mean annual decline: 4.08, 95% CI [-7.44,-0.72], p = 0.02 in ambulant; 4.81, 95% CI [-6.79,-2.82], p < 0.001 in non-ambulant). FVC% captured disease progression in non-ambulant DMD subjects, with an annual loss of 5.47% (95% CI [-6.48,-4.45], p < 0.001). Upper limb function measured with the Performance of Upper Limb (PUL 1.2) showed an annual loss of 4.13 points (95% CI [-4.79,3.47], p < 0.001) in the non-ambulant cohort. Measures of upper limb strength (MyoGrip and MyoPinch) showed a continuous decline independent of the ambulatory status, when reported as percentage predicted (grip force -5.51%, 95% CI [-6.54,-4.48], p < 0.001 in ambulant and a slower decline -2.86%; 95% CI -3.29,-2.43, p < 0.001, in non-ambulant; pinch force: -2.66%, 95% CI [-3.82,-1.51], p < 0.001 in ambulant and -2.23%, 95% CI [-2.92,-1.53], p < 0.001 in non-ambulant). Furthermore, we also explored the novel concept of a composite endpoint by combining respiratory, upper limb function and force domains: we were able to identify clear clinical progression in patients in whom an isolated measurement of only one of these domains failed to appreciate the yearly change. Our study contributes to the field of natural history of DMD, linking the ambulant and non-ambulant phases of the disease, and suggests that composite scores should be explored further.

Cardiac arrhythmia and late-onset muscle weakness caused by a myofibrillar myopathy with unusual histopathological features due to a novel missense mutation in FLNC.

Myofibrillar myopathies (MFM) are mostly adult-onset diseases characterized by progressive morphological alterations of the muscle fibers beginning in the Z-disk and the presence of protein aggregates in the sarcoplasm. They are mostly caused by mutations in different genes that encode Z-disk proteins, including DES, CRYAB, LDB3, MYOT, FLNC and BAG3. A large family of French origin, presenting an autosomal dominant pattern, characterized by cardiac arrhythmia associated to late-onset muscle weakness, was evaluated to clarify clinical, morphological and genetic diagnosis. Muscle weakness began during adult life (over 30 years of age), and had a proximal distribution. Histology showed clear signs of a myofibrillar myopathy, but with unusual, large inclusions. Subsequently, genetic testing was performed in MFM genes available for screening at the time of clinical/histological diagnosis, and desmin (DES), αB-crystallin (CRYAB), myotilin (MYOT) and ZASP (LDB3), were excluded. LMNA gene screening found the p.R296C variant which did not co-segregate with the disease. Genome wide scan revealed linkage to 7q.32, containing the FLNC gene. FLNC direct sequencing revealed a heterozygous c.3646T>A p.Tyr1216Asn change, co-segregating with the disease, in a highly conserved amino acid of the protein. Normal filamin C levels were detected by Western-blot analysis in patient muscle biopsies and expression of the mutant protein in NIH3T3 showed filamin C aggregates. This is an original FLNC mutation in a MFM family with an atypical clinical and histopathological presentation, given the presence of significantly focal lesions and prominent sarcoplasmic masses in muscle biopsies and the constant heart involvement preceding significantly the onset of the myopathy. Though a rare etiology, FLNC gene should not be excluded in early-onset arrhythmia, even in the absence of myopathy, which occurs later in the disease course.

Childhood Pompe disease: clinical spectrum and genotype in 31 patients.

BACKGROUND: As little information is available on children with non-classic presentations of Pompe disease, we wished to gain knowledge of specific clinical characteristics and genotypes. We included all patients younger than 18 years, who had been evaluated at the Pompe Center in Rotterdam, the Netherlands, between 1975 and 2012, excluding those with the classic-infantile form. None were treated with enzyme replacement therapy at the time of evaluation. We collected information on first symptoms, diagnosis, use of a wheelchair and/or respirator, and enzyme and mutation analysis and assessed muscle strength, pulmonary function, and cardiac parameters. RESULTS: Thirty-one patients participated. Median age at symptom onset was 2.6 years (range 0.5-13y) and at diagnosis 4.0 years. Most first problems were delayed motor development and problems related to limb-girdle weakness. Fatigue, persistent diarrhea and problems in raising the head in supine position were other first complaints. Ten patients were asymptomatic at time of diagnosis. Five of them developed symptoms before inclusion in this study. Over 50 % of all patients had low or absent reflexes, a myopathic face, and scoliosis; 29 % were underweight. Muscle strength of the neck flexors, hip extensors, hip flexors, and shoulder abductors were most frequently reduced. Pulmonary function was decreased in over 48 % of the patients; 2 patients had cardiac hypertrophy. Patients with mutations other than the c.-32-13T > G were overall more severely affected, while 18 out of the 21 patients (86 %) with the c.-32-13T > G/'null' genotype were male. CONCLUSIONS: Our study shows that Pompe disease can present with severe mobility and respiratory problems during childhood. Pompe disease should be considered in the differential diagnosis of children with less familiar signs such as disproportional weakness of the neck flexors, unexplained fatigue, persistent diarrhea and unexplained high CK/ASAT/ALAT. Disease presentation appears to be different from adult patients. The majority of affected children with GAA genotype c.-32-13T > G/'null' appeared to be male.

Hammersmith Functional Motor Scale and Motor Function Measure-20 in non ambulant SMA patients.

The aim of this prospective longitudinal multi centric study was to evaluate the correlation between the Hammersmith Functional Motor Scale and the 20 item version of the Motor Function Measure in non ambulant SMA children and adults at baseline and over a 12 month period. Seventy-four non-ambulant patients performed both measures at baseline and 49 also had an assessment 12 month later. At baseline the scores ranged between 0 and 40 on the Hammersmith Motor function Scale and between 3 and 45 on the Motor Function Measure 20. The correlation between the two scales was 0.733. The 12 month changes ranged between -11 and 4 for the Hammersmith and between -11 and 7 for the Motor Function Measure 20. The correlation between changes was 0.48. Our results suggest that both scales provide useful information although they appeared to work differently at the two extremes of the spectrum of abilities. The Hammersmith Motor Function Scale appeared to be more suitable in strong non ambulant patients, while the Motor Function Measures appeared to be more sensitive to capture activities and possible changes in the very weak patients, including more items capturing axial and upper limb activities. The choice of these measures in clinical trials should therefore depend on inclusion criteria and magnitude of expected changes.

Novel TPM3 mutation in a family with cap myopathy and review of the literature.

Cap myopathy is a rare congenital myopathy characterized by the presence of caps within muscle fibres and caused by mutations in ACTA1, TPM2 or TPM3. Thus far, only three cases with TPM3-related cap myopathy have been described. Here, we report on the first autosomal dominant family with cap myopathy in three-generations, caused by a novel heterozygous mutation in the alpha-tropomyosin-slow-encoding gene (TPM3; exon 4; c.445C>A; p.Leu149Ile). The three patients experienced first symptoms of muscle weakness in childhood and followed a slowly progressive course. They presented generalized hypotrophy and mild muscle weakness, elongated face, high arched palate, micrognathia, scoliosis and respiratory involvement. Intrafamilial variability of skeletal deformities, respiratory involvement and mild cardiac abnormalities was noted. Muscle MRI revealed a recognizable pattern of fatty muscle infiltration and masseter muscle hypertrophy. Subsarcolemmal caps were present in 6-10% of the fibres and immunoreactive with anti-tropomyosin antibodies. We conclude that the MRI-pattern of muscle involvement and the presence of masseter muscle hypertrophy in cap myopathy may guide molecular genetic diagnosis towards a mutation in TPM3. Regular respiratory examinations are important, even if patients have no anamnestic clues. We compare our findings to all cases of cap myopathy with identified mutations (n=11), thus far reported in the literature.

Diagnostic criteria for sarcopenia and physical performance.

Relative and absolute muscle mass and muscle strength are used as diagnostic criteria for sarcopenia. We aimed to assess which diagnostic criteria are most associated with physical performance in 180 young (18-30 years) and 281 healthy old participants (69-81 years) of the European study MYOAGE. Diagnostic criteria included relative muscle mass (total or appendicular lean mass (ALM) as percentage of body mass), absolute muscle mass (ALM/height squared and total lean mass), knee extension torque, and handgrip strength. Physical performance comprised walking speed, Timed Up and Go test (TUG), and in a subgroup physical fitness. Diagnostic criteria for sarcopenia and physical performance were standardized, and the associations were analyzed using linear regression models stratified by age category, with adjustments for age, gender, and country. In old participants, relative muscle mass was associated with faster walking speed, faster TUG, and higher physical fitness (all p < 0.001). Absolute muscle mass was not associated with physical performance. Knee extension torque and handgrip strength were associated with faster walking speed (both p ≤ 0.003). Knee extension torque was associated with TUG (p = 0.001). Knee extension torque and handgrip strength were not associated with physical fitness. In young participants, there were no significant associations between diagnostic criteria for sarcopenia and physical performance, except for a positive association between relative muscle mass and physical fitness (p < 0.001). Relative muscle mass, defined as lean mass or ALM percentage, was most associated with physical performance. Absolute muscle mass including ALM/height squared was not associated with physical performance. This should be accounted for when defining sarcopenia.

Diagnostic measures for sarcopenia and bone mineral density.

SUMMARY: Currently used diagnostic measures for sarcopenia utilize different measures of muscle mass, muscle strength, and physical performance. These diagnostic measures associate differently to bone mineral density (BMD), as an example of muscle-related clinical outcome. These differences should be taken into account when studying sarcopenia. INTRODUCTION: Diagnostic measures for sarcopenia utilize different measures of muscle mass, muscle strength, and physical performance. To understand differences between these measures, we determined the association with respect to whole body BMD, as an example of muscle-related clinical outcome. METHODS: In the European cross-sectional study MYOAGE, 178 young (18-30 years) and 274 healthy old participants (69-81 years) were recruited. Body composition and BMD were evaluated using dual-energy X-ray densitometry. Diagnostic measures for sarcopenia were composed of lean mass as percentage of body mass, appendicular lean mass (ALM) as percentage of body mass, ALM divided by height squared (ALM/height(2)), knee extension torque, grip strength, walking speed, and Timed Up and Go test (TUG). Linear regression models were stratified for sex and age and adjusted for age and country, and body composition in separate models. RESULTS: Lean mass and ALM/height(2) were positively associated with BMD (P < 0.001). Significance remained in all sex and age subgroups after further adjustment for fat mass, except in old women. Lean mass percentage and ALM percentage were inversely associated with BMD in old women (P < 0.001). These inverse associations disappeared after adjustment for body mass. Knee extension torque and handgrip strength were positively associated with BMD in all subgroups (P < 0.01), except in old women. Walking speed and TUG were not related to BMD. CONCLUSIONS: The associations between diagnostic measures of sarcopenia and BMD as an example of muscle-related outcome vary widely. Differences between diagnostic measures should be taken into account when studying sarcopenia.

Innovative methods to assess upper limb strength and function in non-ambulant Duchenne patients.

Upper limb assessment in non-ambulant patients remains a challenge. We have designed new tools to precisely assess pinch (MyoPinch), grip (MyoGrip), wrist flexion and extension (MyoWrist) strength. We have also designed a new tool to assess the ability of patients to produce repetitive flexion/extension movements of wrist and fingers (MoviPlate). We have assessed the feasibility and reliability of these new tools in 30 non-ambulant patients with Duchenne muscular dystrophy and in 30 age-matched male controls. Existing measures, such as Motor Function Measure, Tapping, and the Brooke Upper Extremity Functional Rating Scale were also performed. Results demonstrated that assessments were feasible in nearly all upper limbs tested for MyoGrip, MyoPinch and MoviPlate. The reliability of all tests, including MyoWrist which was not feasible in the patients presenting with contractures, was excellent in patients as in controls. Motor capacities decrease with the number of months spent in the wheelchair. The scores in the tests were partially correlated with each other, and with clinical measures such as vital capacity, Motor Function Measure, functional hand scale and Brooke score. This study validates a panel of upper limb muscle strength and function measures for Duchenne Muscular Dystrophy which can be applied from controls to extremely weak patients.

Familial reducing body myopathy with cytoplasmic bodies and rigid spine revisited: identification of a second LIM domain mutation in FHL1.

OBJECTIVE: Reducing body myopathy (RBM) is a rare progressive disorder of muscle characterized by intracytoplasmic inclusions, which stain strongly with menadione-NBT (nitroblue tetrazolium). We recently identified the four and a half LIM domain gene FHL1 located on chromosome Xq26 as the causative gene for RBM. So far eight familial cases and 21 sporadic patients with RBM have been reported in the literature. METHODS: We ascertained a total of 8 members of a German family initially reported by Goebel et al. as a mixed myopathy with rigid spine myopathy and reducing as well as cytoplasmic bodies. Clinical findings in the original and additional family members have been reviewed. Mutation detection was performed by direct sequencing of FHL1 exons. RESULTS: We identified a novel mutation (p.C150R) in the second LIM domain of FHL1 in six family members (1 male, 5 females). The male index patient was the most affected member presenting with rigid spine, followed by rapidly progressive muscle weakness. He died from the consequences of respiratory insufficiency at the age of 29.5 years. His sister, mother, grandmother, aunt and female cousin all carried the mutation in the heterozygous state. The sister is clinically unaffected; their mother had myopathic changes in her muscle biopsy, while the grandmother showed first signs of weakness at 50 years of age. The 54-year-old aunt and her daughter are clinically asymptomatic. CONCLUSION: We report a novel LIM2 domain mutation in FHL1 in a previously reported family with RBM with cytoplasmic bodies and spinal rigidity. While the male index patient was significantly affected, female carriers show varying manifestations and may be asymptomatic, likely reflecting varying degrees of X-inactivation. RBM continues to be associated with mutations in the LIM2 domain of FHL1. We also confirm our earlier observation that mutations at the N-terminal end of the LIM2 domain seem to be milder compared to mutations seen at the C-terminal part of the domain which cause severe disease even in female carriers.

Differentiating Emery-Dreifuss muscular dystrophy and collagen VI-related myopathies using a specific CT scanner pattern.

Bethlem myopathy and Ullrich congenital muscular dystrophy are part of the heterogeneous group of collagen VI-related muscle disorders. They are caused by mutations in collagen VI (ColVI) genes (COL6A1, COL6A2, and COL6A3) while LMNA mutations cause autosomal dominant Emery-Dreifuss muscular dystrophy. A muscular dystrophy pattern and contractures are found in all three conditions, making differential diagnosis difficult especially in young patients when cardiomyopathy is absent. We retrospectively assessed upper and lower limb muscle CT scans in 14 Bethlem/Ullrich patients and 13 Emery-Dreifuss patients with identified mutations. CT was able to differentiate Emery-Dreifuss muscular dystrophy from ColVI-related myopathies in selected thigh muscles and to a lesser extent calves muscles: rectus femoris fatty infiltration was selectively present in Bethlem/Ullrich patients while posterior thigh muscles infiltration was more prominently found in Emery-Dreifuss patients. A more severe fatty infiltration particularly in the leg posterior compartment was found in the Emery-Dreifuss group.

Correlation of enzyme activity and clinical phenotype in POMT1-associated dystroglycanopathies.

BACKGROUND: Mutations in protein O-mannosyltransferases (POMTs) cause a heterogeneous group of muscular dystrophies with abnormal glycosylation of alpha-dystroglycan (dystroglycanopathies). The wide spectrum of clinical severities ranges from Walker-Warburg syndrome (WWS), associated with brain and eye abnormalities, to mild forms of limb girdle muscular dystrophy (LGMD). OBJECTIVE: The aim of this study was to elucidate the impact of mutations in POMT1 on the clinical phenotype. METHODS: We examined 2 patients with POMT1-associated alpha-dystroglycanopathy, 1 displaying a LGMD2K and 1 with a WWS phenotype. Using dermal fibroblasts, we analyzed the influence of the POMT1 mutations on the glycosylation status of alpha-dystroglycan, protein O-mannosyltransferase activity, and the stability of the mutant POMT1 protein. RESULTS: We report on novel compound heterozygous mutations in POMT1 (p.L171A and p.A589VfsX38) that result in LGMD2K. We further demonstrate that a homozygous splice site mutation of a recently identified WWS patient results in POMT1 p.del77-93. Using dermal fibroblasts, we show that mannosyltransferase activity is reduced in the patients and that stability of POMT1 mutant proteins p.A589VfsX38 and p.del77-93 is significantly decreased. CONCLUSIONS: Our results suggest that dermal fibroblasts can be applied to facilitate the diagnostic analysis of dystroglycanopathy patients as well as to study the pathogenic mechanism of POMT mutations. Characterization of the POMT1 substrate protein alpha-dystroglycan and POMT in vitro mannosyltransferase activity shows that the severity of the clinical phenotype of the patients analyzed is inversely correlated with POMT activity.

Electron microscopy in myofibrillar myopathies reveals clues to the mutated gene.

We studied the ultrastructural characteristics in patients with myofibrillar myopathy (MFM) and differentiated between MFM-subtypes using electron microscopic (EM) findings. The ultrastructural findings in 19 patients with different genetically proven MFMs (9 desmin, 5 alphaB-crystallin, 3 ZASP, 2 myotilin) were analyzed. In one ZASPopathy, we additionally performed an immunoEM study, using antibodies against desmin, alphaB-crystallin, ZASP and myotilin. The ultrastructural findings in desminopathies and alphaB-crystallinopathies were very similar and consisted of electrondense granulofilamentous accumulations and sandwich formations. They differed in the obvious presence of early apoptotic nuclear changes in alphaB-crystallinopathies. ZASPopathies were characterized by filamentous bundles (labeled with the myotilin antibody on immunoEM), and floccular accumulations of thin filamentous material. Tubulofilamentous inclusions in sarcoplasm and myonuclei in combination with filamentous bundles were characteristic for myotilinopathies. We conclude that MFMs ultrastructural findings can direct diagnostic efforts towards the causal gene mutated, and that EM should be included in the diagnostic workup of MFMs.

Non-invasive ventilation reduces respiratory tract infections in children with neuromuscular disorders.

Non-invasive ventilation (NIV) has been shown to improve gas exchange and survival in patients with chronic neuromuscular disorders. Little is known about its influence on respiratory tract infections (RTIs). Twenty-four patients with regular use of NIV and 11 patients without NIV with neuromuscular disorders answered a questionnaire concerning the use of NIV and assisted coughing techniques, the status of influenza and pneumococcus vaccination, and the frequency and severity of RTIs. Additionally, we performed a retrospective chart review of twelve patients who were ventilated over a period of at least 5 years. In the first year of NIV consultations of a general practitioner due to RTI decreased from 9.2+/-20.8 to 3.2+/-5.3 per year (P<0.005), the number of antibiotic treatment due to RTI decreased from 4.1+/-3.4 to 1.9+/-2.2 per year (P<0.005) and the number of hospital admissions due to RTI decreased from 1.6+/-1.7 to 0.7+/-1.3 per year (P<0.005). Vaccinations against influenza and/or pneumococcus did not have a significant influence on the rate of infections. In 12 patients using NIV for more than 5 years the incidence of RTI requiring hospital admission decreased from 0.54+/-0.41/year in the pre-ventilation period to 0.12+/-0.09/year in the NIV period (P<0.005). NIV had a favorable impact on respiratory infectious complications in children with neuromuscular disorders.

Nemaline myopathy with exclusively intranuclear rods and a novel mutation in ACTA1 (Q139H).

Nemaline myopathies (NM) are a rare group of muscle disorders, but represent one of the most common forms of congenital myopathy. The clinical picture ranges from severe muscular hypotonia often leading to death during childhood to mild forms with long life expectancy. Diagnosis is made by muscle biopsy showing characteristic sarcoplasmic and sometimes intranuclear rod bodies. So far, disease-associated mutations have been detected in six genes without any simple correlation between genotype and phenotype or histological findings. We report a patient with a phenotype typical of congenital onset nemaline myopathy and exclusively intranuclear rods. Mutation analysis revealed a new heterozygous missense mutation in exon 3 of the ACTA1 gene (Q139H). Molecular modelling predicts that substitution of Q139 for H139 alters the amino acid side chains and hydrogen bonding which may alter the nucleotide binding cleft by adding 'bulk' to the mutated molecule. Two-dimensional gel electrophoresis demonstrates that mutant actin Q139H is expressed at approximately half the level of wild-type actin in the patient's muscle. We speculate that these alterations, although not directly affecting the nuclear export signal, negatively interfere with the nuclear export of the mutated protein and thereby cause retention of mutant actin and intranuclear rod formation.

Genetic analysis of the first 4 patients with beta-ureidopropionase deficiency.

beta-Ureidopropionase is the third enzyme of the pyrimidine degradation pathway and it catalyses the irreversible hydrolysis of N-carbamyl-ss-aminoisobutyric acid or N-carbamyl-ss-alanine to beta-aminoisobutyric acid or ss-alanine, ammonia, and CO2. Analysis of the beta-ureidopropionase gene (UPB1) of the first 4 patients presenting with a complete enzyme deficiency, revealed the presence of 2 splice-site mutations (IVS1-2A>G and IVS8-1G>A) and one missense mutation (A85E). RT-PCR analysis of the complete beta-ureidopropionase cDNA suggested that both splice-site mutations lead to a variety of alternative splice variants, with deletions of a single or several exons. The alanine at position 85 was not conserved in other eukaryotic beta-ureidopropionase protein sequences.