Form Of Nemaline Myopathy Research Articles

Actin Polymerization Defects Induce Mitochondrial Dysfunction in Cellular Models of Nemaline Myopathies.

Nemaline myopathy (NM) is one of the most common forms of congenital myopathy and it is identified by the presence of "nemaline bodies" (rods) in muscle fibers by histopathological examination. The most common forms of NM are caused by mutations in the Actin Alpha 1 (ACTA1) and Nebulin (NEB) genes. Clinical features include hypotonia and muscle weakness. Unfortunately, there is no curative treatment and the pathogenetic mechanisms remain unclear. In this manuscript, we examined the pathophysiological alterations in NM using dermal fibroblasts derived from patients with mutations in ACTA1 and NEB genes. Patients' fibroblasts were stained with rhodamine-phalloidin to analyze the polymerization of actin filaments by fluorescence microscopy. We found that patients' fibroblasts showed incorrect actin filament polymerization compared to control fibroblasts. Actin filament polymerization defects were associated with mitochondrial dysfunction. Furthermore, we identified two mitochondrial-boosting compounds, linoleic acid (LA) and L-carnitine (LCAR), that improved the formation of actin filaments in mutant fibroblasts and corrected mitochondrial bioenergetics. Our results indicate that cellular models can be useful to study the pathophysiological mechanisms involved in NM and to find new potential therapies. Furthermore, targeting mitochondrial dysfunction with LA and LCAR can revert the pathological alterations in NM cellular models.

P.02 Mild nemaline myopathy 10 caused by a novel missense homozygous mutation in LMOD3: broadening the phenotype-genotype correlation

Nemaline myopathy (NM) is a heterogeneous group of conditions characterized by the presence of muscle weakness and the accumulation of nemaline bodies in the muscle biopsy. So far, mutations in 14 genes have been related to both recessive and dominant forms of NM. <i>LMOD3</i> encodes for the Leimodin-3 protein, which is expressed in the skeletal and cardiac muscle. The majority of described mutations in <i>LMOD3</i> are truncating mutations and cause a severe autosomal recessive congenital myopathy (NEM-10), characterized by early-onset hypotonia, generalized muscle weakness and respiratory insufficiency. Most of the patients also present polyhydramnios, reduced fetal movements, arthrogryposis or frequent contractures, and they usually die during early childhood. Here we describe a family with two affected adult sisters with a novel homozygous missense mutation in LMOD3 presenting a very mild phenotype, providing further evidence to the phenotype-genotype correlation of NEM-10. The proband, a 54-year-old woman was the first child of healthy consanguineous parents. She presented with mild hypotonia at birth and delayed motor milestones. She was never able to run, and presented frequent falls during infancy. Examination revealed high arched palate, bilateral facial weakness, and mild weakness involving neck flexors, iliopsoas and knee flexors. Her sister had the same phenotype. Serum CK were within normal levels. A muscle biopsy taken from the triceps brachii revealed mild variation in the fiber size, and very rare fibres containing nemaline bodies. Both affected sisters presented a novel homozygous missense c.1030C>T, p.Arg344Trp (NM_198271.4) mutation identified in the <i>LMOD3</i> gene through a NGS custom gene panel analyzing 139 neuromuscular-related genes. Our work broadens the mutational spectrum of <i>LMOD3</i> gene and contributes to better understand the clinical heterogeneity of nemaline myopathy.

Acute and chronic tirasemtiv treatment improves in vivo and in vitro muscle performance in actin-based nemaline myopathy mice.

Nemaline myopathy, a disease of the actin-based thin filament, is one of the most frequent congenital myopathies. To date, no specific therapy is available to treat muscle weakness in nemaline myopathy. We tested the ability of tirasemtiv, a fast skeletal troponin activator that targets the thin filament, to augment muscle force—both in vivo and in vitro—in a nemaline myopathy mouse model with a mutation (H40Y) in Acta1. In Acta1H40Y mice, treatment with tirasemtiv increased the force response of muscles to submaximal stimulation frequencies. This resulted in a reduced energetic cost of force generation, which increases the force production during a fatigue protocol. The inotropic effects of tirasemtiv were present in locomotor muscles and, albeit to a lesser extent, in respiratory muscles, and they persisted during chronic treatment, an important finding as respiratory failure is the main cause of death in patients with congenital myopathy. Finally, translational studies on permeabilized muscle fibers isolated from a biopsy of a patient with the ACTA1H40Y mutation revealed that at physiological Ca2+ concentrations, tirasemtiv increased force generation to values that were close to those generated in muscle fibers of healthy subjects. These findings indicate the therapeutic potential of fast skeletal muscle troponin activators to improve muscle function in nemaline myopathy due to the ACTA1H40Y mutation, and future studies should assess their merit for other forms of nemaline myopathy and for other congenital myopathies.

A recurrent copy number variation of the NEB triplicate region: only revealed by the targeted nemaline myopathy CGH array.

Recently, new large variants have been identified in the nebulin gene (NEB) causing nemaline myopathy (NM). NM constitutes a heterogeneous group of disorders among the congenital myopathies, and disease-causing variants in NEB are a main cause of the recessively inherited form of NM. NEB consists of 183 exons and it includes homologous sequences such as a 32-kb triplicate region (TRI), where eight exons are repeated three times (exons 82-89, 90-97, 98-105). In human, the normal copy number of NEB TRI is six (three copies in each allele). Recently, we described a custom NM-CGH microarray designed to detect copy number variations (CNVs) in the known NM genes. The array has now been updated to include all the currently known 10 NM genes. The NM-CGH array is superior in detecting CNVs, especially of the NEB TRI, that is not included in the exome capture kits. To date, we have studied 266 samples from 196 NM families using the NM-CGH microarray, and identified a novel recurrent NEB TRI variation in 13% (26/196) of the families and in 10% of the controls (6/60). An analysis of the breakpoints revealed adjacent repeat elements, which are known to predispose for rearrangements such as CNVs. The control CNV samples deviate only one copy from the normal six copies, whereas the NM samples include CNVs of up to four additional copies. Based on this study, NEB seems to tolerate deviations of one TRI copy, whereas addition of two or more copies might be pathogenic.

Characterization of a Kbtbd13‐Deficient Mouse Model That Recapitulates Nemaline Myopathy With Mutations in The KBTBD13 Gene

BackgroundNemaline myopathy (NM) is the most common non‐dystrophic congenital myopathy and is characterized by muscle weakness. Recently a novel implicated gene was discovered ‐ KBTBD13 – of which the function of its protein product is unknown. NM patients with KBTBD13 mutations have muscle weakness and show a typical slowness of movement. Here, we generated a mouse model that lacks the kbtbd13 gene to obtain insight into the role of this new gene and its protein product and to elucidate the pathophysiology of this new form of NM.MethodsKbtbd13‐KO mice are viable and live into adulthood. Muscle weights of cardiac and skeletal muscle were collected. Intact soleus muscles of 3 month‐old male kbtbd13‐KO and wild‐type (WT) mice were used for in vitro experiments. Muscle tendons were attached to a force transducer and a length motor. Muscles were electrically activated by field stimulation at 30°C.ResultsMaximal tetanic force at 150Hz was reduced in kbtbd13‐KO (n=10). The relative force‐frequency relationship in kbtbd13‐KO mice was shifted to the left compared to WT mice. Both activation and relaxation kinetics were slower in kbtbd13‐KO mice. Muscle weight data reveal atrophy of skeletal muscles, whereas the cardiac muscle shows hypertrophy of the left ventricle.ConclusionKbtbd13‐KO mice phenocopy the normal life span and typical muscle weakness observed in NM patients with mutations in KBTBD13. Hence, we present a novel NM mouse model that is a useful tool to elucidate the role of this new gene and its protein product and to study the mechanism underlying muscle weakness observed in patients.

G.P.274: Cold shock domain protein A – A novel nemaline myopathy-causing gene?

Mutation analysis of the known nemaline myopathy (NM)-causing genes in a Finnish patient with an unusual form of NM, with legs clearly stronger than arms, did not reveal the cause of the disease. A heterozygous TPM3 splice site mutation, delTAGG, in intron 1 was identified, inherited from the healthy father. The mutation was predicted to be recessive, but no TPM3 mutation was found on the maternal allele. Large copy number variations affecting the known nine NM genes were excluded by NM-CGH array analysis. Whole-exome sequencing revealed a heterozygous RYR1 missense mutation, p.Leu2031Phe, predicted to be probably damaging by PolyPhen-2, but the healthy father also carried this mutation. No other putative pathogenic mutation in RYR1 was found in the patient, and in myoblasts of the patient, epigenetic silencing of the maternal allele was ruled out. Exome sequencing did, however, disclose two putative pathogenic mutations in the cold shock domain protein A encoding gene, CSDA . The CSDA gene has 11 exons and encodes three different protein isoforms: 303 aa, 372 aa and 200 aa. CSDA is highly expressed in skeletal muscle and heart. Our patient was compound heterozygous for two missense mutations, p.Ser34Arg and p.Arg129Trp, in CSDA, each inherited from one of the healthy parents. The mutations are not listed in the EVS database (http://evs.gs.washington.edu/EVS/). Ser34 and Arg129 are highly conserved between species, and reside in the DNA-binding domain (cold shock domain) of CSDA. Furthermore, Ser34 is phosphorylated by ERK2 and GSK3 β , and the phosphorylation is thought to be important for the formation of nuclear CSDA complexes and binding of CSDA to single-stranded DNA. Western blot analysis of cultured myotubes from the patient confirms CSDA expression on the protein level. Further analyses are ongoing in order to quantify the expression levels and determine the intracellular location of CSDA in myoblasts from the patient and healthy controls.

G.P.262: Selectivity patterns on lower limb skeletal muscle imaging in patients with nemaline myopathy

Nemaline myopathy (NM) is a clinically and genetically heterogeneous skeletal muscle disorder. An increasing number of reports have shown the usefulness of skeletal muscle imaging for differentiating neuromuscular disorders. However, the characteristic patterns in most cases do not correspond with the typical selectivity of muscle involvement in NM. In this study, we aimed to clarify the relationship between genotype and imaging phenotype in NM. Twelve patients (median age, 4.8years) who visited the Department of Pediatric Neurology at NCNP hospital and were diagnosed with NM by muscle biopsy between 2005 and 2013 were studied. The direct sequence of ACTA1 was initially analyzed in all patients. Then, a next generation sequence was performed for the other cases detected no mutations in ACTA1. Magnetic resonance imaging (MRI) of the lower limb skeletal muscles was performed before muscle biopsy in all patients. We classified the patients into 3 groups according to the region of selective involvement on MRI. Group 1, soleus and gastrocnemius muscles (<i>n</i>=5; 3 NEB mutations, 1 ACTA1 mutation, 1 TNNT3 mutation,) Group 2, vastus lateralis, vastus medialis, and semimembranosus muscles, with relative sparing of the rectus femoris, sartorius, gracilis, adductor longus, and semitendinousus muscles (<i>n</i>=3; 2 NEB mutations, 1 unknown mutation), and Group 3, other patterns (<i>n</i>=4; 2 ACTA1 mutations, 2 unknown mutations). All patients in Group 1 could walk unassisted and had no mental retardation. The Group 2 patients could not walk for a long distance. In Group 3, the clinical spectrum ranged from mild to severe. The selectivity pattern in Group 1 was previously reported for a mild form of NM with an NEB mutation. However, the pattern observed in Group 2 has not been reported, suggesting that NM due to NEB mutations shows variable patterns of selectivity.

A Novel Form of Nemaline Myopathy Caused by an In-Frame Deletion in TNNT1 (P5.095)

A Novel Form of Nemaline Myopathy Caused by an In-Frame Deletion in <i>TNNT1</i> (P5.095)

Multimodal MRI and 31P-MRS Investigations of the ACTA1(Asp286Gly) Mouse Model of Nemaline Myopathy Provide Evidence of Impaired In Vivo Muscle Function, Altered Muscle Structure and Disturbed Energy Metabolism

Nemaline myopathy (NM), the most common non-dystrophic congenital disease of skeletal muscle, can be caused by mutations in the skeletal muscle α-actin gene (ACTA1) (~25% of all NM cases and up to 50% of severe forms of NM). Muscle function of the recently generated transgenic mouse model carrying the human Asp286Gly mutation in the ACTA1 gene (Tg(ACTA1)Asp286Gly) has been mainly investigated in vitro. Therefore, we aimed at providing a comprehensive picture of the in vivo hindlimb muscle function of Tg(ACTA1)Asp286Gly mice by combining strictly noninvasive investigations. Skeletal muscle anatomy (hindlimb muscles, intramuscular fat volumes) and microstructure were studied using multimodal magnetic resonance imaging (Dixon, T2, Diffusion Tensor Imaging [DTI]). Energy metabolism was studied using 31-phosphorus Magnetic Resonance Spectroscopy (31P-MRS). Skeletal muscle contractile performance was investigated while applying a force-frequency protocol (1–150 Hz) and a fatigue protocol (6 min–1.7 Hz). Tg(ACTA1)Asp286Gly mice showed a mild muscle weakness as illustrated by the reduction of both absolute (30%) and specific (15%) maximal force production. Dixon MRI did not show discernable fatty infiltration in Tg(ACTA1)Asp286Gly mice indicating that this mouse model does not reproduce human MRI findings. Increased T2 values were observed in Tg(ACTA1)Asp286Gly mice and might reflect the occurrence of muscle degeneration/regeneration process. Interestingly, T2 values were linearly related to muscle weakness. DTI experiments indicated lower λ2 and λ3 values in Tg(ACTA1)Asp286Gly mice, which might be associated to muscle atrophy and/or the presence of histological anomalies. Finally 31P-MRS investigations illustrated an increased anaerobic energy cost of contraction in Tg(ACTA1)Asp286Gly mice, which might be ascribed to contractile and non-contractile processes. Overall, we provide a unique set of information about the anatomic, metabolic and functional consequences of the Asp286Gly mutation that might be considered as relevant biomarkers for monitoring the severity and/or the progression of NM and for assessing the efficacy of potential therapeutic interventions.

Combined MRI and 31P-MRS Investigations of the ACTA1(H40Y) Mouse Model of Nemaline Myopathy Show Impaired Muscle Function and Altered Energy Metabolism

Nemaline myopathy (NM) is the most common disease entity among non-dystrophic skeletal muscle congenital diseases. Mutations in the skeletal muscle α-actin gene (ACTA1) account for ∼25% of all NM cases and are the most frequent cause of severe forms of NM. So far, the mechanisms underlying muscle weakness in NM patients remain unclear. Additionally, recent Magnetic Resonance Imaging (MRI) studies reported a progressive fatty infiltration of skeletal muscle with a specific muscle involvement in patients with ACTA1 mutations. We investigated strictly noninvasively the gastrocnemius muscle function of a mouse model carrying a mutation in the ACTA1 gene (H40Y). Skeletal muscle anatomy (hindlimb muscles and fat volumes) and energy metabolism were studied using MRI and 31Phosphorus magnetic resonance spectroscopy. Skeletal muscle contractile performance was investigated while applying a force-frequency protocol (from 1–150 Hz) and a fatigue protocol (80 stimuli at 40 Hz). H40Y mice showed a reduction of both absolute (−40%) and specific (−25%) maximal force production as compared to controls. Interestingly, muscle weakness was associated with an improved resistance to fatigue (+40%) and an increased energy cost. On the contrary, the force frequency relationship was not modified in H40Y mice and the extent of fatty infiltration was minor and not different from the WT group. We concluded that the H40Y mouse model does not reproduce human MRI findings but shows a severe muscle weakness which might be related to an alteration of intrinsic muscular properties. The increased energy cost in H40Y mice might be related to either an impaired mitochondrial function or an alteration at the cross-bridges level. Overall, we provided a unique set of anatomic, metabolic and functional biomarkers that might be relevant for monitoring the progression of NM disease but also for assessing the efficacy of potential therapeutic interventions at a preclinical level.

Clinical and pathologic analysis of an autosomal recessive kindred with nemaline myopathy

To investigate the clinical, pathological and ultrastructural features of nemaline myopathy (NM). The clinical manifestations of four patients in a rare autosomal recessive kindred with nemaline myopathy were analyzed retrospectively. Biopsied specimens of left gastrocnemius from the proband were detected and observed through light microscope using enzymatic histochemical methods for histopathological changes and through electron microscope for ultrastructural features. Four affected siblings in this kindred had an onset at birth or fetal stage, among whom two case were respiratory independent with delayed attainment of motor milestones and general muscle atrophy complying with typical form of NM, whereas the other two did not achieve adequate spontaneous movement or breathing, and died at neonatal period according with severe form of NM. Other clinical characteristics of elongated face, tent-shaped mouth and high-arched palate were also found. The proband had normal serum muscle enzymes and the karyotypic analysis showed a normal G band. Brain magnetic resonance image (MRI) indicated no abnormality. Electromyogram (EMG) showed typical muscle-derived damages of biceps, triceps, brachioradial muscle, vastus medialis muscle, anterior tibial muscle and gastrocnemius with normal motor conduction velocity of bilateral tibial nerves and common peroneal nerves. Myofibrillar atrophy was found through light microscopy with fiber type 1 predominance shown by ATP enzyme staining, yet without indication of lipid or glycogen deposition by ORO or PAS staining. Modified gomori trichrome (MGT) treatment resulted in dark-red staining of nemaline bodies in myofibril cytoplasm. And it was also observed as purple-red staining followed by hematoxylin-eosin (HE) treatment. Electron microscopic observation by lead-uranium double staining showed widened interstitial myofibrils, focal myofilament disorganization, partial myofilament atrophy, focal dissolution or necrosis, partial myofibrils nucleus pyknosis, numerous subsarcolemmal and perinuclear rod-like structures, partial nemaline bodies connected with Z discs, and mitochondria vacuolation or disappearance. NM is among congenital myopathies characterized by marked clinical and pathological heterogeneity. The diagnosis of NM should be based upon typical clinical and histopathological features.

Nemaline myopathy: clinical, histochemical and immunohistochemical features

Nemaline myopathy (NM) is a congenital disease that leads to hypotonia and feeding difficulties in neonates. Some cases have a more benign course, with skeletal abnormalities later in life. We analyzed a series of eight patients with NM obtained from a retrospective analysis of 4300 muscle biopsies. Patients were classified as having the typical form in five cases, intermediate form in two cases and severe form in one case. Histochemical analysis showed mixed rods distribution in all cases and predominance of type I fibers in five cases. Immunohistochemical analysis showed abnormal nebulin expression in all patients (four heterogeneous and four absent), homogeneous desmin expression in four cases, strongly positive in three and absent in one, fast myosin expression in a mosaic pattern in six cases and absent in two cases. There was no specific relation between these protein expression patterns and the clinical forms of NM.

Heterogeneity of nemaline myopathy cases with skeletal muscle α‐actin gene mutations

Nemaline myopathy (NM) is the most common of several congenital myopathies that present with skeletal muscle weakness and hypotonia. It is clinically heterogeneous and the diagnosis is confirmed by identification of nemaline bodies in affected muscles. The skeletal muscle alpha-actin gene (ACTA1) is one of five genes for thin filament proteins identified so far as responsible for different forms of NM. We have screened the ACTA1 gene in a cohort of 109 unrelated patients with NM. Here, we describe clinical and pathological features associated with 29 ACTA1 mutations found in 38 individuals from 28 families. Although ACTA1 mutations cause a remarkably heterogeneous range of phenotypes, they were preferentially associated with severe clinical presentations (p < 0.0001). Most pathogenic ACTA1 mutations were missense changes with two instances of single base pair deletions. Most patients with ACTA1 mutations had no prior family history of neuromuscular disease (24/28). One severe case, caused by compound heterozygous recessive ACTA1 mutations, demonstrated increased alpha-cardiac actin expression, suggesting that cardiac actin might partially compensate for ACTA1 abnormalities in the fetal/neonatal period. This cohort also includes the first instance of an ACTA1 mutation manifesting with adult-onset disease and two pedigrees exhibiting potential incomplete penetrance. Overall, ACTA1 mutations are a common cause of NM, accounting for more than half of severe cases and 26% of all NM cases in this series.

Nebulin mutations in autosomal recessive nemaline myopathy: an update

We report mutational analysis of the last 42 exons of the nebulin gene ( NEB) in 77 patients with various forms of nemaline myopathy. In addition to the previously described six mutations in five families, we identified 12 novel recessive mutations in 13 families. Affected individuals were homozygous for the mutations in five families and compound heterozygous in two, while in the remaining cases only one heterozygous mutation was identified. The majority of the mutations were frameshifts due to small deletions or insertions; also common were point mutations causing premature stop codons or abnormal splicing, while missense mutations appeared rare. There were no obvious mutational hotspots, although four unrelated patients showed mutations in the differentially expressed exon 177d, and another three showed mutations in exon 184. Most of the mutations are predicted to result in truncated or internally deleted proteins. Mutations in the differentially expressed exons are expected to reduce the nebulin isoform diversity necessary for normal muscle development.

An autosomal dominant congenital myopathy with cores and rods is associated with a neomutation in the RYR1 gene encoding the skeletal muscle ryanodine receptor.

Central core disease (CCD) and nemaline myopathy (NM) are congenital myopathies for which differential diagnosis is often based on the presence either of cores or rods. Missense mutations in the skeletal muscle ryanodine receptor gene (RYR1) have been identified in some families with CCD. Mutations in the alpha-tropomyosin and alpha-actin genes have been associated with most dominant forms of NM. Analysis of the RYR1 cDNA in a French family identified a novel Y4796C mutation that lies in the C-terminal channel-forming domain of the RyR1 protein. This mutation was linked not only to a severe and penetrant form of CCD, but also to the presence of rods in the muscle fibres and to the malignant hyperthermia susceptibility (MHS) phenotype. The Y4796C mutation was introduced into a rabbit RYR1 cDNA and expressed in HEK-293 cells. Expression of the mutant RYR1 cDNA produced channels with increased caffeine sensitivity and a significantly reduced maximal level of Ca(2+) release. Single-cell Ca(2+) analysis showed that the resting cytoplasmic level was increased by 60% in cells expressing the mutant channel. These data support the view that the rate of Ca(2+) leakage is increased in the mutant channel. The resulting chronic elevation in myoplasmic concentration is likely to be responsible for the severe expression of the disease. Haplotyping analysis indicated that the mutation arose as a neomutation in the proband. This first report of a neomutation in the RYR1 gene has strong implications for genetic linkage studies of MHS or CCD, two diseases characterized by a genetic heterogeneity.

A female patient with the severe infantile form of nemaline myopathy

A female patient with the severe infantile form of nemaline myopathy

Nemaline myopathy: Comparative muscle histochemistry in the severe neonatal, moderate congenital, and adult-onset forms

A histochemical study of biopsied muscle specimens from patients with the 3 forms of nemaline myopathy (i.e., severe neonatal, moderate congenital, and adult-onset), classified on the basis of clinical symptoms, was conducted. A close relationship could not be found between the number of rods and the severity of weakness in any form. Type 1 fiber atrophy and predominance or type 2B fiber deficiency was the usual finding in all forms. In the moderate congenital form, type 1 fiber atrophy and predominance became more apparent in patients with a protracted course, suggesting that the histochemical abnormalities are progressive. The abnormal fiber type distribution is assumed to be related to the pathogenetic mechanism of nemaline myopathy in all forms. Because acid phosphatase activity was increased in muscle fibers of patients with rapid progression, an autodegenerative process inducing lysosomal enzyme activation may be responsible for the acute clinical progression and muscle fiber loss in this disorder.

Neonatal nemaline myopathy presenting with multiple joint contractures

A sporadic case of the rare and most severe neonatal form of nemaline myopathy is reported. Intrauterine manifestation included reduced fetal movements and breech position with a normal amount of amniotic fluid. After delivery by Caesarian section at 34 weeks of gestation the infant boy, who was not asphyctic, failed to establish spontaneous breathing and required immediate intubation and ventilation. Marked muscular hypotonia and weakness persisted and reflexes remained absent. Hip dislocation, joint contractures, absent palmar creases, prominent lateral palatal ridges and cryptorchidism were interpreted as consequent to prenatal paralysis. The boy died after 5 h due to hyaline membrane disease and meconium aspiration. At autopsy the skeletal muscles were found to be hypoplastic. The muscle fibres contained numerous rods, a typical finding of nemaline myopathy. Nemaline myopathy should be considered in fetuses and newborns with multiple joint contractures, severe muscular weakness and respiratory insufficiency.