A recurrent ACTA1 amino acid change in mosaic form causes milder asymmetric myopathy.

We describe three patients with asymmetric congenital myopathy without definite nemaline bodies and one patient with severe nemaline myopathy. In all four patients, the phenotype had been caused by pathogenic missense variants in ACTA1 leading to the same amino acid change, p.(Gly247Arg). The three patients with milder myopathy were mosaic for their variants. In contrast, in the severely affected patient, the missense variant was present in a de novo, constitutional form. The grade of mosaicism in the three mosaic patients ranged between 20 % and 40 %. We speculate that the milder clinical and histological manifestations of the same ACTA1 variant in the patients with mosaicism reflect the lower abundance of mutant actin in their muscle tissue. Similarly, the asymmetry of body growth and muscle weakness may be a consequence of the affected cells being unevenly distributed. The partial improvement in muscle strength with age in patients with mosaicism might be due to an increased proportion over time of nuclei carrying and expressing two normal alleles.

NEB mutations disrupt the super-relaxed state of myosin and remodel the muscle metabolic proteome in nemaline myopathy.

Nemaline myopathy (NM) is one of the most common non-dystrophic genetic muscle disorders. NM is often associated with mutations in the NEB gene. Even though the exact NEB-NM pathophysiological mechanisms remain unclear, histological analyses of patients' muscle biopsies often reveal unexplained accumulation of glycogen and abnormally shaped mitochondria. Hence, the aim of the present study was to define the exact molecular and cellular cascade of events that would lead to potential changes in muscle energetics in NEB-NM. For that, we applied a wide range of biophysical and cell biology assays on skeletal muscle fibres from NM patients as well as untargeted proteomics analyses on isolated myofibres from a muscle-specific nebulin-deficient mouse model. Unexpectedly, we found that the myosin stabilizing conformational state, known as super-relaxed state, was significantly impaired, inducing an increase in the energy (ATP) consumption of resting muscle fibres from NEB-NM patients when compared with controls or with other forms of genetic/rare, acquired NM. This destabilization of the myosin super-relaxed state had dynamic consequences as we observed a remodeling of the metabolic proteome in muscle fibres from nebulin-deficient mice. Altogether, our findings explain some of the hitherto obscure hallmarks of NM, including the appearance of abnormal energy proteins and suggest potential beneficial effects of drugs targeting myosin activity/conformations for NEB-NM.

Comprehensive transcriptomic analysis shows disturbed calcium homeostasis and deregulation of T lymphocyte apoptosis in inclusion body myositis.

OBJECTIVE: Inclusion body myositis (IBM) has an unclear molecular etiology exhibiting both characteristic inflammatory T-cell activity and rimmed-vacuolar degeneration of muscle fibers. Using in-depth gene expression and splicing studies, we aimed at understanding the different components of the molecular pathomechanisms in IBM. METHODS: We performed RNA-seq on RNA extracted from skeletal muscle biopsies of clinically and histopathologically defined IBM (n = 24), tibial muscular dystrophy (n = 6), and histopathologically normal group (n = 9). In a comprehensive transcriptomics analysis, we analyzed the differential gene expression, differential splicing and exon usage, downstream pathway analysis, and the interplay between coding and non-coding RNAs (micro RNAs and long non-coding RNAs). RESULTS: We observe dysregulation of genes involved in calcium homeostasis, particularly affecting the T-cell activity and regulation, causing disturbed Ca2+-induced apoptotic pathways of T cells in IBM muscles. Additionally, LCK/p56, which is an essential gene in regulating the fate of T-cell apoptosis, shows increased expression and altered splicing usage in IBM muscles. INTERPRETATION: Our analysis provides a novel understanding of the molecular mechanisms in IBM by showing a detailed dysregulation of genes involved in calcium homeostasis and its effect on T-cell functioning in IBM muscles. Loss of T-cell regulation is hypothesized to be involved in the consistent observation of no response to immune therapies in IBM patients. Our results show that loss of apoptotic control of cytotoxic T cells could indeed be one component of their abnormal cytolytic activity in IBM muscles.

Out-of-Frame Mutations in ACTN2 Last Exon Cause a Dominant Distal Myopathy With Facial Weakness.

BACKGROUND AND OBJECTIVES: To clinically, genetically, and histopathologically characterize patients presenting with an unusual combination of distal myopathy and facial weakness, without involvement of upper limb or shoulder girdle muscles. METHODS: Two families with a novel form of actininopathy were identified. Patients had been followed up over 10 years. Their molecular genetic diagnosis was not clear after extensive investigations, including analysis of candidate genes and FSHD1-related D4Z4 repeats. RESULTS: Patients shared a similar clinical phenotype and a common pattern of muscle involvement. They presented with a very slowly progressive myopathy involving anterior lower leg and facial muscles. Muscle MRI finding showed complete fat replacement of anterolateral compartment muscles of the lower legs with variable involvement of soleus and gastrocnemius but sparing thigh muscles. Muscle biopsy showed internalized nuclei, myofibrillar disorganization, and rimmed vacuoles. High-throughput sequencing identified in each proband a heterozygous single nucleotide deletion (c.2558del and c.2567del) in the last exon of the ACTN2 gene. The deletions are predicted to lead to a novel but unstructured slightly extended C-terminal amino acid sequence. DISCUSSION: Our findings indicate an unusual form of actininopathy with specific molecular and clinical features. Actininopathy should be considered in the differential diagnosis of distal myopathy combined with facial weakness.

Missense mutations in small muscle protein X-linked (SMPX) cause distal myopathy with protein inclusions.

Using deep phenotyping and high-throughput sequencing, we have identified a novel type of distal myopathy caused by mutations in the Small muscle protein X-linked (SMPX) gene. Four different missense mutations were identified in ten patients from nine families in five different countries, suggesting that this disease could be prevalent in other populations as well. Haplotype analysis of patients with similar ancestry revealed two different founder mutations in Southern Europe and France, indicating that the prevalence in these populations may be higher. In our study all patients presented with highly similar clinical features: adult-onset, usually distal more than proximal limb muscle weakness, slowly progressing over decades with preserved walking. Lower limb muscle imaging showed a characteristic pattern of muscle involvement and fatty degeneration. Histopathological and electron microscopic analysis of patient muscle biopsies revealed myopathic findings with rimmed vacuoles and the presence of sarcoplasmic inclusions, some with amyloid-like characteristics. In silico predictions and subsequent cell culture studies showed that the missense mutations increase aggregation propensity of the SMPX protein. In cell culture studies, overexpressed SMPX localized to stress granules and slowed down their clearance.

A novel COL6A2 mutation causing late-onset limb-girdle muscular dystrophy.

Limb-girdle muscular dystrophies (LGMD) are genetic disorders characterized by weakness of predominantly proximal limb and trunk muscles due to progressive loss of muscle tissue. Collagen VI-related muscular dystrophies usually display more generalized muscle involvement combined with contractures and/or hyperlaxity of distal finger joints. LGMD-like phenotype of collagenopathy has only rarely been described and as reported is usually of childhood onset. We identified a Finnish family with COL6A2-related LGMD with autosomal dominant inheritance and very late onset at 40-60 years of age. Since the mutation was previously unreported, the pathognomonic findings on muscle MRI were the decisive clue for the correct diagnosis.

An unusual ryanodine receptor 1 (RYR1) phenotype: Mild calf-predominant myopathy.

OBJECTIVE: To identify the genetic defect causing a distal calf myopathy with cores. METHODS: Families with a genetically undetermined calf-predominant myopathy underwent detailed clinical evaluation, including EMG/nerve conduction studies, muscle biopsy, laboratory investigations, and muscle MRI. Next-generation sequencing and targeted Sanger sequencing were used to identify the causative genetic defect in each family. RESULTS: A novel deletion-insertion mutation in ryanodine receptor 1 (RYR1) was found in the proband of the index family and segregated with the disease in 6 affected relatives. Subsequently, we found 2 more families with a similar calf-predominant myopathy segregating with unique RYR1-mutated alleles. All patients showed a very slowly progressive myopathy without episodes of malignant hyperthermia or rhabdomyolysis. Muscle biopsy showed cores or core-like changes in all families. CONCLUSIONS: Our findings expand the spectrum of RYR1-related disorders to include a calf-predominant myopathy with core pathology and autosomal dominant inheritance. Two families had unique and previously unreported RYR1 mutations, while affected persons in the third family carried 2 previously known mutations in the same dominant allele.

Dominantly inherited distal nemaline/cap myopathy caused by a large deletion in the nebulin gene.

We report the first family with a dominantly inherited mutation of the nebulin gene (NEB). This ∼100 kb in-frame deletion encompasses NEB exons 14-89, causing distal nemaline/cap myopathy in a three-generation family. It is the largest deletion characterized in NEB hitherto. The mutated allele was shown to be expressed at the mRNA level and furthermore, for the first time, a deletion was shown to cause the production of a smaller mutant nebulin protein. Thus, we suggest that this novel mutant nebulin protein has a dominant-negative effect, explaining the first documented dominant inheritance of nebulin-caused myopathy. The index patient, a young man, was more severely affected than his mother and grandmother. His first symptom was foot drop at the age of three, followed by distal muscle atrophy, slight hypomimia, high-arched palate, and weakness of the neck and elbow flexors, hands, tibialis anterior and toe extensors. Muscle biopsies showed myopathic features with type 1 fibre predominance in the index patient and nemaline bodies and cap-like structures in biopsies from his mother and grandmother. The muscle biopsy findings constitute a further example of nemaline bodies and cap-like structures being part of the same spectrum of pathological changes.

Recessive PYROXD1 mutations cause adult-onset limb-girdle-type muscular dystrophy.

OBJECTIVE: To describe adult-onset limb-girdle-type muscular dystrophy caused by biallelic variants in the PYROXD1 gene, which has been recently linked to early-onset congenital myofibrillar myopathy. METHODS: Whole exome sequencing was performed for adult-onset neuromuscular disease patients with no molecular diagnosis. Patients with PYROXD1 variants underwent clinical characterization, lower limb muscle MRI, muscle biopsy and spirometry. A yeast complementation assay was used to determine the biochemical consequences of the genetic variants. RESULTS: We identified four patients with biallelic PYROXD1 variants. Three patients, who had symptom onset in their 20s or 30s, were homozygous for the previously described p.Asn155Ser. The fourth patient, with symptom onset at age 49, was compound heterozygous for p.Asn155Ser variant and previously unknown p.Tyr354Cys. All patients presented with a LGMD-type phenotype of symmetric muscle weakness and wasting. Symptoms started in proximal muscles of the lower limbs, and progressed slowly to involve also upper limbs in a proximal-predominant fashion. All patients remained ambulant past the age of 60. They had restrictive lung disease but no cardiac impairment. Muscle MRI showed strong involvement of anterolateral thigh muscles. Muscle biopsy displayed chronic myopathic changes. Yeast complementation assay demonstrated the p.Tyr354Cys mutation to impair PYROXD1 oxidoreductase ability. CONCLUSION: PYROXD1 variants can cause an adult-onset slowly progressive LGMD-type phenotype.

Homozygous Nonsense Mutation p.Q274X in TRIM63 (MuRF1) in a Patient with Mild Skeletal Myopathy and Cardiac Hypertrophy.

TRIM63 mutations have been described as a potential cause for cardiac and skeletal myopathy in only one family so far. We describe a new patient carrying the same homozygous TRIM63 nonsense mutation c.739 C>T p.Q247X, that was originally reported in two members of a Spanish family manifesting cardiac hypertrophy. One of these original patients also had an additional heterozygous mutation in TRIM54 and a much more severe phenotype also involving skeletal muscles, and a digenic inheritance was therefore suggested. Our case report confirms the role of TRIM63 as a new cardiac myopathy gene, although it is unclear whether the homozygous p.Q247X mutation alone is sufficient to cause an additional skeletal myopathy.

The complexity of titin splicing pattern in human adult skeletal muscles.

BACKGROUND: Mutations in the titin gene (TTN) cause a large spectrum of diseases affecting skeletal and/or cardiac muscle. TTN includes 363 coding exons, a repeated region with a high degree of complexity, isoform-specific elements, and metatranscript-only exons thought to be expressed only during fetal development. Although three main classes of isoforms have been described so far, alternative splicing events (ASEs) in different tissues or in different developmental and physiological states have been reported. METHODS: To achieve a comprehensive view of titin ASEs in adult human skeletal muscles, we performed a RNA-Sequencing experiment on 42 human biopsies collected from 12 anatomically different skeletal muscles of 11 individuals without any skeletal-muscle disorders. RESULTS: We confirmed that the skeletal muscle N2A isoforms are highly prevalent, but we found an elevated number of alternative splicing events, some at a very high level. These include previously unknown exon skipping events and alternative 5' and 3' splice sites. Our data suggests the partial inclusion in the TTN transcript of some metatranscript-only exons and the partial exclusion of canonical N2A exons. CONCLUSIONS: This study provides an extensive picture of the complex TTN splicing pattern in human adult skeletal muscle, which is crucial for a proper clinical interpretation of TTN variants.

Cranioplasty with Adipose-Derived Stem Cells, Beta-Tricalcium Phosphate Granules and Supporting Mesh: Six-Year Clinical Follow-Up Results.

Several alternative techniques exist to reconstruct skull defects. The complication rate of the cranioplasty procedure is high and the search for optimal materials and techniques continues. To report long-term results of patients who have received a cranioplasty using autologous adipose-derived stem cells (ASCs) seeded on beta-tricalcium phosphate (betaTCP) granules. Between 10/2008 and 3/2010, five cranioplasties were performed (four females, one male; average age 62.0 years) using ASCs, betaTCP granules and titanium or resorbable meshes. The average defect size was 8.1 × 6.7 cm2 . Patients were followed both clinically and radiologically. The initial results were promising, with no serious complications. Nevertheless, in the long-term follow-up, three of the five patients were re-operated due to graft related problems. Two patients showed marked resorption of the graft, which led to revision surgery. One patient developed a late infection (7.3 years post-operative) that required revision surgery and removal of the graft. One patient had a successfully ossified graft, but was re-operated due to recurrence of the meningioma 2.2 years post-operatively. One patient had an uneventful clinical follow-up, and the cosmetic result is satisfactory, even though skull x-rays show hypodensity in the borders of the graft. Albeit no serious adverse events occurred, the 6-year follow-up results of the five cases are unsatisfactory. The clinical results are not superior to results achieved by conventional cranial repair methods. The use of stem cells in combination with betaTCP granules and supporting meshes in cranial defect reconstruction need to be studied further before continuing with clinical trials. Stem Cells Translational Medicine 2017;6:1576-1582.

Distinct Muscle Biopsy Findings in Genetically Defined Adult-Onset Motor Neuron Disorders.

The objective of this study was to characterize and compare muscle histopathological findings in 3 different genetic motor neuron disorders. We retrospectively re-assessed muscle biopsy findings in 23 patients with autosomal dominant lower motor neuron disease caused by p.G66V mutation in CHCHD10 (SMAJ), 10 X-linked spinal and bulbar muscular atrophy (SBMA) and 11 autosomal dominant c9orf72-mutated amyotrophic lateral sclerosis (c9ALS) patients. Distinct large fiber type grouping consisting of non-atrophic type IIA muscle fibers were 100% specific for the late-onset spinal muscular atrophies (SMAJ and SBMA) and were never observed in c9ALS. Common, but less specific findings included small groups of highly atrophic rounded type IIA fibers in SMAJ/SBMA, whereas in c9ALS, small group atrophies consisting of small-caliber angular fibers involving both fiber types were more characteristic. We also show that in the 2 slowly progressive motor neuron disorders (SMAJ and SBMA) the initial neurogenic features are often confused with considerable secondary "myopathic" changes at later disease stages, such as rimmed vacuoles, myofibrillar aggregates and numerous fibers reactive for fetal myosin heavy chain (dMyHC) antibodies. Based on our findings, muscle biopsy may be valuable in the diagnostic work-up of suspected motor neuron disorders in order to avoid a false ALS diagnosis in patients without clear findings of upper motor neuron lesions.

Myxoinflammatory fibroblastic sarcoma of the nose: First reported case at an unusual location (nasal dorsum), with a review of the literature.

Myxoinflammatory fibroblastic sarcoma (MIFS) is a rare, low-grade, malignant, soft-tissue tumor that typically affects the distal extremities of middle-aged patients. In most cases, it presents as a painless, slowly growing mass within the subcutaneous tissue. It is associated with a low rate of metastasis but a high rate of local recurrence. In addition to the distal extremities, MIFS has been reported in the thigh, arm, forearm, groin, upper back, neck, and temporal area. As far as we know, no case has been previously reported in the nasal area. We report for the first time a case of MIFS presenting on the dorsum of the nose. The painless, 3.0-cm tumor was initially mistaken for reticular erythematous mucinosis, a benign skin condition that occurs when fibroblasts produce abnormally large amounts of mucopolysaccharides. The tumor was surgically removed in its entirety with surgical margins of 3 to 5 mm. During 4 years of follow-up, no clinical or radiologic evidence of a recurrence or metastasis was seen. We discuss the imaging and histologic features of MIFS, as well as its clinical management and follow-up, and we review related reports in the literature.

Diagnostically important muscle pathology in DNAJB6 mutated LGMD1D.

INTRODUCTION: Limb girdle muscular dystrophies are a large group of both dominantly and recessively inherited muscle diseases. LGMD1D is caused by mutated DNAJB6 and the molecular pathogenesis is mediated by defective chaperonal function leading to impaired handling of misfolded proteins which normally would be degraded. Here we aim to clarify muscle pathology of LGMD1D in order to facilitate diagnostic accuracy. After following six Finnish LGMD1D families, we analysed 21 muscle biopsies obtained from 15 patients at different time points after the onset of symptoms. All biopsies were obtained from the lower limb muscles and processed for routine histochemistry, extensive immunohistochemistry and electron microscopy. RESULTS: Histopathological findings were myopathic or dystrophic combined with rimmed vacuolar pathology, and small myofibrillar aggregates. These myofibrillar inclusions contained abnormal accumulation of a number of proteins such as myotilin, αB-crystallin and desmin on immunohistochemistry, and showed extensive myofibrillar disorganization with excess of Z-disk material on ultrastructure. Later in the disease process the rimmed vacuolar pathology dominated with rare cases of pronounced larger pleomorphic myofibrillar aggregates. The rimmed vacuoles were reactive for several markers of defect autophagy such as ubiquitin, TDP-43, p62 and SMI-31. CONCLUSIONS: Since DNAJB6 is known to interact with members of the chaperone assisted selective autophagy complex (CASA), including BAG3 - a known myofibrillar myopathy causing gene, the molecular muscle pathology is apparently mediated through impaired functions of CASA and possibly other complexes needed for the maintenance of the Z-disk and sarcomeric structures. The corresponding findings on histopathology offer clues for the diagnosis.

Mutations in HSPB8 causing a new phenotype of distal myopathy and motor neuropathy.

OBJECTIVE: To report novel disease and pathology due to HSPB8 mutations in 2 families with autosomal dominant distal neuromuscular disease showing both myofibrillar and rimmed vacuolar myopathy together with neurogenic changes. METHODS: We performed whole-exome sequencing (WES) in tandem with linkage analysis and candidate gene approach as well as targeted next-generation sequencing (tNGS) to identify causative mutations in 2 families with dominant rimmed vacuolar myopathy and a motor neuropathy. Pathogenic variants and familial segregation were confirmed using Sanger sequencing. RESULTS: WES and tNGS identified a heterozygous change in HSPB8 in both families: c.421A > G p.K141E in family 1 and c.151insC p.P173SfsX43 in family 2. Affected patients had a distal myopathy that showed myofibrillar aggregates and rimmed vacuoles combined with a clear neurogenic component both on biopsy and neurophysiologic studies. MRI of lower limb muscles demonstrated diffuse tissue changes early in the disease stage progressing later to fatty replacement typical of a myopathy. CONCLUSION: We expand the understanding of disease mechanisms, tissue involvement, and phenotypic outcome of HSPB8 mutations. HSPB8 is part of the chaperone-assisted selective autophagy (CASA) complex previously only associated with Charcot-Marie-Tooth type 2L (OMIM 60673) and distal hereditary motor neuronopathy type IIa. However, we now demonstrate that patients can develop a myopathy with histologic features of myofibrillar myopathy with aggregates and rimmed vacuoles, similar to the pathology in myopathies due to gene defects in other compounds of the CASA complex such as BAG3 and DNAJB6 after developing the early neurogenic effects.

Differential isoform expression and selective muscle involvement in muscular dystrophies.

Despite the expression of the mutated gene in all muscles, selective muscles are involved in genetic muscular dystrophies. Different muscular dystrophies show characteristic patterns of fatty degenerative changes by muscle imaging, even to the extent that the patterns have been used for diagnostic purposes. However, the underlying molecular mechanisms explaining the selective involvement of muscles are not known. To test the hypothesis that different muscles may express variable amounts of different isoforms of muscle genes, we applied a custom-designed exon microarray containing probes for 57 muscle-specific genes to assay the transcriptional profiles in sets of human adult lower limb skeletal muscles. Quantitative real-time PCR and whole transcriptome sequencing were used to further analyze the results. Our results demonstrate significant variations in isoform and gene expression levels in anatomically different muscles. Comparison of the known patterns of selective involvement of certain muscles in two autosomal dominant titinopathies and one autosomal dominant myosinopathy, with the isoform and gene expression results, shows a correlation between the specific muscles involved and significant differences in the level of expression of the affected gene and exons in these same muscles compared with some other selected muscles. Our results suggest that differential expression levels of muscle genes and isoforms are one determinant in the selectivity of muscle involvement in muscular dystrophies.

No cardiomyopathy in X-linked myopathy with excessive autophagy.

In X-linked myopathy with excessive autophagy (XMEA) progressive sarcoplasmic accumulation of autolysosomes filled with undegraded debris leads to atrophy and weakness of skeletal muscles. XMEA is caused by compromised acidification of lysosomes resulting from hypofunction of the proton pump vacuolar ATPase (V-ATPase), due to hypomorphic mutations in VMA21, whose protein product assembles V-ATPase. To what extent the cardiac muscle is affected is unknown. Therefore we performed a comprehensive cardiac evaluation in four male XMEA patients, and also examined pathology of one deceased patient's cardiac and skeletal muscle. None of the symptomatic men (aged 25-48 years) had history or symptoms of cardiomyopathy. Resting electrocardiograms and echocardiographies were normal. MRI showed normal left ventricle ejection fraction and myocardial mass. Myocardial late-gadolinium enhancement was not detected. The deceased patient's skeletal but not cardiac muscle showed characteristic accumulation of autophagic vacuoles. In conclusion, in classic XMEA the myocardium is structurally, electrically and clinically spared.