Mutations In TPM2 Research Articles

Linking the Glu150Ala mutation in slow skeletal muscle Tpm3.12 to congenital myopathy

Linking the Glu150Ala mutation in slow skeletal muscle Tpm3.12 to congenital myopathy

Troponin and a Myopathy-Linked Mutation in TPM3 Inhibit Cofilin-2-Induced Thin Filament Depolymerization.

Uniform actin filament length is required for synchronized contraction of skeletal muscle. In myopathies linked to mutations in tropomyosin (Tpm) genes, irregular thin filaments are a common feature, which may result from defects in length maintenance mechanisms. The current work investigated the effects of the myopathy-causing p.R91C variant in Tpm3.12, a tropomyosin isoform expressed in slow-twitch muscle fibers, on the regulation of actin severing and depolymerization by cofilin-2. The affinity of cofilin-2 for F-actin was not significantly changed by either Tpm3.12 or Tpm3.12-R91C, though it increased two-fold in the presence of troponin (without Ca2+). Saturation of the filament with cofilin-2 removed both Tpm variants from the filament, although Tpm3.12-R91C was more resistant. In the presence of troponin (±Ca2+), Tpm remained on the filament, even at high cofilin-2 concentrations. Both Tpm3.12 variants inhibited filament severing and depolymerization by cofilin-2. However, the inhibition was more efficient in the presence of Tpm3.12-R91C, indicating that the pathogenic variant impaired cofilin-2-dependent actin filament turnover. Troponin (±Ca2+) further inhibited but did not completely stop cofilin-2-dependent actin severing and depolymerization.

A novel variant in the tropomyosin 3 gene presenting as an adult-onset distal myopathy - a case report

BackgroundWe report a patient with a novel c.737 C > T variant (p.Ser246Leu) of the TPM3 gene presenting with adult-onset distal myopathy.Case presentationA 35-year-old Chinese male patient presentedwith a history of progressive finger weakness. Physical examination revealeddifferential finger extension weakness, together with predominant fingerabduction, elbow flexion, ankle dorsiflexion and toe extension weakness. MuscleMRI showed disproportionate fatty infiltration of the glutei, sartorius andextensor digitorum longus muscles without significant wasting. Muscle biopsy andultrastructural examination showed a non-specific myopathic pattern withoutnemaline or cap inclusions. Genetic sequencing revealed a novel heterozygousp.Ser246Leu variant (c.737C>T) of the TPM3 gene which is predicted tobe pathogenic. This variant is located in the area of the TPM3 genewhere the protein product interacts with actin at position Asp25 of actin.Mutations of TPM3 in these loci have been shown to alter the sensitivityof thin filaments to the influx of calcium ions.ConclusionThis report further expands the phenotypicspectrum of myopathies associated with TPM3 mutations, as mutations in TPM3had not previously been reported with adult-onset distal myopathy. We also discuss the interpretation of variantsof unknown significance in patients with TPM3 mutations and summarisethe typical muscle MRI findings of patients with TPM3 mutations.

Molecular Mechanisms of Deregulation of Muscle Contractility Caused by the R168H Mutation in TPM3 and Its Attenuation by Therapeutic Agents

The substitution for Arg168His (R168H) in γ-tropomyosin (TPM3 gene, Tpm3.12 isoform) is associated with congenital muscle fiber type disproportion (CFTD) and muscle weakness. It is still unclear what molecular mechanisms underlie the muscle dysfunction seen in CFTD. The aim of this work was to study the effect of the R168H mutation in Tpm3.12 on the critical conformational changes that myosin, actin, troponin, and tropomyosin undergo during the ATPase cycle. We used polarized fluorescence microscopy and ghost muscle fibers containing regulated thin filaments and myosin heads (myosin subfragment-1) modified with the 1,5-IAEDANS fluorescent probe. Analysis of the data obtained revealed that a sequential interdependent conformational-functional rearrangement of tropomyosin, actin and myosin heads takes place when modeling the ATPase cycle in the presence of wild-type tropomyosin. A multistep shift of the tropomyosin strands from the outer to the inner domain of actin occurs during the transition from weak to strong binding of myosin to actin. Each tropomyosin position determines the corresponding balance between switched-on and switched-off actin monomers and between the strongly and weakly bound myosin heads. At low Ca2+, the R168H mutation was shown to switch some extra actin monomers on and increase the persistence length of tropomyosin, demonstrating the freezing of the R168HTpm strands close to the open position and disruption of the regulatory function of troponin. Instead of reducing the formation of strong bonds between myosin heads and F-actin, troponin activated it. However, at high Ca2+, troponin decreased the amount of strongly bound myosin heads instead of promoting their formation. Abnormally high sensitivity of thin filaments to Ca2+, inhibition of muscle fiber relaxation due to the appearance of the myosin heads strongly associated with F-actin, and distinct activation of the contractile system at submaximal concentrations of Ca2+ can lead to muscle inefficiency and weakness. Modulators of troponin (tirasemtiv and epigallocatechin-3-gallate) and myosin (omecamtiv mecarbil and 2,3-butanedione monoxime) have been shown to more or less attenuate the negative effects of the tropomyosin R168H mutant. Tirasemtiv and epigallocatechin-3-gallate may be used to prevent muscle dysfunction.

O.01 Correlating molecular genetic testing for rare genetic variants with a broad clinicopathologic spectrum of congenital myopathies

Congenital myopathies (CMs) are a group of clinically heterogeneous disorders presenting with a range of overlapping findings, making diagnosis challenging. Despite advances in genetic testing, many cases remain undiagnosed. The Beggs' laboratory CM cohort, which currently totals over 1,100 individuals, constitutes a robust dataset of existing cases. Subjects were ascertained based on clinical presentation and pathologic diagnoses suggestive of CM. We report that over 65% have been solved by a combination of single gene, panel, or genomic sequencing, and/or RNA-Seq. Of 727 solved cases, 610 had mutations in either ACTA1, DNM2, MTM1, NEB, RYR1, SELENON, TPM2, TPM3, or TTN. All these genes are well-known causes of congenital myopathy. While all patients shared a myopathic pattern of weakness and hypotonia, a wide spectrum of clinicopathologic features were appreciated among the cases. The remaining 117 cases had mutations in one of 52 different genes, including a number of rare or novel genes, as well as some more commonly mutated non-CM disease genes in patients who presented atypically for those associated conditions. Among a heterogeneous group of 96 probands with congenital myopathy with nonspecific or no pathology, 31 (32%) had SELENON mutations; 9 (9.4%) had RYR1 mutations and 6 (6.25%) had TTN mutations. This survey of the Beggs' laboratory cohort cases, accumulated over roughly 20 years, emphasizes the crucial and growing role of WES/WGS in establishing a molecular genetic diagnosis of CM, as opposed to disease-based gene panels or single-gene testing, which may miss cases where the presentation does not fit the classical description of the disorder, or where multiple genes may present with overlapping clinicopathologic diagnoses. Moreover, certain syndromes not known to present predominantly with myopathic symptoms may go undiagnosed if genetic testing is done in a more targeted fashion based on specific clinical symptoms or pathologic diagnosis alone.

Combining Gene Mutation with Expression of Candidate Genes to Improve Diagnosis of Escobar Syndrome.

Escobar syndrome is a rare, autosomal recessive disorder that affects the musculoskeletal system and the skin. Mutations in the CHRNG and TPM2 genes are associated with this pathology. In this study, we conducted a clinical and genetic investigation of five patients and also explored via in silico and gene expression analysis their phenotypic variability. In detail, we identified a patient with a novel composite heterozygous variant of the CHRNG gene and two recurrent mutations in both CHRNG and TPM2 in the rest of the patients. As for the clinical particularities, we reported a list of modifier genes in a patient suffering from myopathy. Moreover, we identified decreased expression of IGF-1, which could be related to the short stature of Escobar patients, and increased expression of POLG1 specific to patients with TPM2 mutation. Through this study, we identified the genetic spectrum of Escobar syndrome in the Tunisian population, which will allow setting up genetic counseling and prenatal diagnosis for families at risk. In addition, we highlighted relevant biomarkers that could differentiate between patients with different genetic defects.

Molecular Mechanisms of the Deregulation of Muscle Contraction Induced by the R90P Mutation in Tpm3.12 and the Weakening of This Effect by BDM and W7.

Point mutations in the genes encoding the skeletal muscle isoforms of tropomyosin can cause a range of muscle diseases. The amino acid substitution of Arg for Pro residue in the 90th position (R90P) in γ-tropomyosin (Tpm3.12) is associated with congenital fiber type disproportion and muscle weakness. The molecular mechanisms underlying muscle dysfunction in this disease remain unclear. Here, we observed that this mutation causes an abnormally high Ca2+-sensitivity of myofilaments in vitro and in muscle fibers. To determine the critical conformational changes that myosin, actin, and tropomyosin undergo during the ATPase cycle and the alterations in these changes caused by R90P replacement in Tpm3.12, we used polarized fluorimetry. It was shown that the R90P mutation inhibits the ability of tropomyosin to shift towards the outer domains of actin, which is accompanied by the almost complete depression of troponin’s ability to switch actin monomers off and to reduce the amount of the myosin heads weakly bound to F-actin at a low Ca2+. These changes in the behavior of tropomyosin and the troponin–tropomyosin complex, as well as in the balance of strongly and weakly bound myosin heads in the ATPase cycle may underlie the occurrence of both abnormally high Ca2+-sensitivity and muscle weakness. BDM, an inhibitor of myosin ATPase activity, and W7, a troponin C antagonist, restore the ability of tropomyosin for Ca2+-dependent movement and the ability of the troponin–tropomyosin complex to switch actin monomers off, demonstrating a weakening of the damaging effect of the R90P mutation on muscle contractility.

A Cross-Sectional Study of Nemaline Myopathy.

ObjectiveNemaline myopathy (NM) is a rare neuromuscular condition with clinical and genetic heterogeneity. To establish disease natural history, we performed a cross-sectional study of NM, complemented by longitudinal assessment and exploration of pilot outcome measures.MethodsFifty-seven individuals with NM were recruited at 2 family workshops, including 16 examined at both time points. Participants were evaluated by clinical history and physical examination. Functional outcome measures included the Motor Function Measure (MFM), pulmonary function tests (PFTs), myometry, goniometry, and bulbar assessments.ResultsThe most common clinical classification was typical congenital (54%), whereas 42% had more severe presentations. Fifty-eight percent of individuals needed mechanical support, with 26% requiring wheelchair, tracheostomy, and feeding tube. The MFM scale was performed in 44 of 57 participants and showed reduced scores in most with little floor/ceiling effect. Of the 27 individuals completing PFTs, abnormal values were observed in 65%. Last, bulbar function was abnormal in all patients examined, as determined with a novel outcome measure. Genotypes included mutations in ACTA1 (18), NEB (20), and TPM2 (2). Seventeen individuals were genetically unresolved. Patients with pathogenic ACTA1 and NEB variants were largely similar in clinical phenotype. Patients without genetic resolution had more severe disease.ConclusionWe present a comprehensive cross-sectional study of NM. Our data identify significant disabilities and support a relatively stable disease course. We identify a need for further diagnostic investigation for the genetically unresolved group. MFM, PFTs, and the slurp test were identified as promising outcome measures for future clinical trials.

Molecular Mechanisms of Muscle Weakness Associated with E173A Mutation in Tpm3.12. Troponin Ca2+ Sensitivity Inhibitor W7 Can Reduce the Damaging Effect of This Mutation.

Substitution of Ala for Glu residue in position 173 of γ-tropomyosin (Tpm3.12) is associated with muscle weakness. Here we observe that this mutation increases myofilament Ca2+-sensitivity and inhibits in vitro actin-activated ATPase activity of myosin subfragment-1 at high Ca2+. In order to determine the critical conformational changes in myosin, actin and tropomyosin caused by the mutation, we used the technique of polarized fluorimetry. It was found that this mutation changes the spatial arrangement of actin monomers and myosin heads, and the position of the mutant tropomyosin on the thin filaments in muscle fibres at various mimicked stages of the ATPase cycle. At low Ca2+ the E173A mutant tropomyosin shifts towards the inner domains of actin at all stages of the cycle, and this is accompanied by an increase in the number of switched-on actin monomers and myosin heads strongly bound to F-actin even at relaxation. Contrarily, at high Ca2+ the amount of the strongly bound myosin heads slightly decreases. These changes in the balance of the strongly bound myosin heads in the ATPase cycle may underlie the occurrence of muscle weakness. W7, an inhibitor of troponin Ca2+-sensitivity, restores the increase in the number of myosin heads strongly bound to F-actin at high Ca2+ and stops their strong binding at relaxation, suggesting the possibility of using Ca2+-desensitizers to reduce the damaging effect of the E173A mutation on muscle fibre contractility.

A recurrent pathogenic variant in TPM2 reveals further phenotypic and genetic heterogeneity in multiple pterygium syndrome-related disorders.

Multiple pterygium syndrome (MPS) disorders are a phenotypically and genetically heterogeneous group of conditions characterized by multiple joint contractures (arthrogryposis), pterygia (joint webbing) and other developmental defects. MPS is most frequently inherited in an autosomal recessive fashion but X-linked and autosomal dominant forms also occur. Advances in genomic technologies have identified many genetic causes of MPS-related disorders and genetic diagnosis requires large targeted next generation sequencing gene panels or genome-wide sequencing approaches. Using the Illumina TruSightOne clinical exome assay, we identified a recurrent heterozygous missense substitution in TPM2 (encoding beta tropomyosin) in three unrelated individuals. This was confirmed to have arisen as a de novo event in the two patients with parental samples. TPM2 mutations have previously been described in association with a variety of dominantly inherited neuromuscular phenotypes including nemaline myopathy, congenital fibre-type disproportion, distal arthrogryposis and trismus pseudocamptodactyly, and in a patient with autosomal recessive Escobar syndrome and a nemaline myopathy. The three cases reported here had overlapping but variable features. Our findings expand the range of TMP2-related phenotypes and indicate that de novo TMP2 mutations should be considered in isolated cases of MPS-related conditions.

Thin filament dysfunctions caused by mutations in tropomyosin Tpm3.12 and Tpm1.1

Tropomyosin is the major regulator of the thin filament. In striated muscle its function is to bind troponin complex and control the access of myosin heads to actin in a Ca2+-dependent manner. It also participates in the maintenance of thin filament length by regulation of tropomodulin and leiomodin, the pointed end-binding proteins. Because the size of the overlap between actin and myosin filaments affects the number of myosin heads which interact with actin, the filament length is one of the determinants of force development. Numerous point mutations in genes encoding tropomyosin lead to single amino acid substitutions along the entire length of the coiled coil that are associated with various types of cardiomyopathy and skeletal muscle disease. Specific regions of tropomyosin interact with different binding partners; therefore, the mutations affect diverse tropomyosin functions. In this review, results of studies on mutations in the genes TPM1 and TPM3, encoding Tpm1.1 and Tpm3.12, are described. The paper is particularly focused on mutation-dependent alterations in the mechanisms of actin-myosin interactions and dynamics of the thin filament at the pointed end.

The Primary Causes of Muscle Dysfunction Associated with the Point Mutations in Tpm3.12; Conformational Analysis of Mutant Proteins as a Tool for Classification of Myopathies.

Point mutations in genes encoding isoforms of skeletal muscle tropomyosin may cause nemaline myopathy, cap myopathy (Cap), congenital fiber-type disproportion (CFTD), and distal arthrogryposis. The molecular mechanisms of muscle dysfunction in these diseases remain unclear. We studied the effect of the E173A, R90P, E150A, and A155T myopathy-causing substitutions in γ-tropomyosin (Tpm3.12) on the position of tropomyosin in thin filaments, and the conformational state of actin monomers and myosin heads at different stages of the ATPase cycle using polarized fluorescence microscopy. The E173A, R90P, and E150A mutations produced abnormally large displacement of tropomyosin to the inner domains of actin and an increase in the number of myosin heads in strong-binding state at low and high Ca2+, which is characteristic of CFTD. On the contrary, the A155T mutation caused a decrease in the amount of such heads at high Ca2+ which is typical for mutations associated with Cap. An increase in the number of the myosin heads in strong-binding state at low Ca2+ was observed for all mutations associated with high Ca2+-sensitivity. Comparison between the typical conformational changes in mutant proteins associated with different myopathies observed with α-, β-, and γ-tropomyosins demonstrated the possibility of using such changes as tests for identifying the diseases.

Novel mutations in TPM2 and PIEZO2 are responsible for distal arthrogryposis (DA) 2B and mild DA in two Chinese families

BackgroundDistal arthrogryposis (DA) is a group of clinically and genetically heterogeneous disorders that involve multiple congenital limb contractures and comprise at least 10 clinical subtypes. Here, we describe our findings in two Chinese families: Family 1 with DA2B (MIM 601680) and Family 2 with mild DA.MethodsTo map the disease locus, two-point linkage analysis was performed with microsatellite markers closed to TPM2, TNNI2/TNNT3 and TNNC2. In Family 1, a positive LOD (logarithm of odds) score was only obtained at the microsatellite marker close to TPM2 and mutation screening was performed using direct sequencing of TPM2 in the proband. In Family 2, for the LOD score that did not favor linkage to any markers, whole-exome sequencing (WES) was performed on the proband. PCR–restriction fragment length polymorphism (RFLP) and bioinformatics analysis were then applied to identify the pathogenic mutations in two families. In order to correlate genotype with phenotype in DA, retrospective analyses of phenotypic features according to the TPM2 and PIEZO2 mutation spectrums were carried out.ResultsA heterozygous missense mutation c.308A > G (p.Q103R) in TPM2 in Family 1, and a novel variation c.8153G > A (p.R2718Q) in PIEZO2 in Family 2 were identified. Each of the two novel variants was co-segregated with the DA manifestations in the corresponding family. Bioinformatics analysis from several tools supported the pathogenicity of the mutations. Furthermore, our study suggests that there is no relation between the types or locations of TPM2 mutations and the clinical characteristics, and that different inheritance modes and mutation types concerning PIEZO2 cause distinct clinical manifestations.ConclusionsWe report two novel mutations within TPM2 and PIEZO2 responsible for DA2B and mild DA in two Chinese families, respectively. Our study expands the spectrum of causal mutations in the TPM2 and PIEZO2 genes.

CONGENITAL MYOPATHIES: NEMALINE AND TITINOPATHIES: P.237Congenital fatal cap-rod myopathy due to a de novo autosomal dominant pathogenic ACTA1 variant

Cap disease is a rare structural congenital myopathy (CM) associated with hypotonia, proximal and facial muscle weakness, and frequently scoliosis and respiratory involvement. Mutations in TPM2, TPM3 and ACTA1 have been associated with cap disease, as well as nemaline myopathy. Combined caps and nemaline rods have been reported in the same patient due to a mutation in TPM3. Here, we report the first case of a severe, fatal CM with distinct and separate caps and nemaline rods identified in skeletal muscle biopsies. The patient was born at 37 weeks of gestation, with a history of polyhydramnios, little spontaneous movements at birth, generalised hypotonia, and required immediate ventilatory support. He died 20 days after birth. Ante mortem and post mortem biopsies from the quadriceps, and post mortem biopsies from the biceps and diaphragm showed diffuse fibre hypotrophy without specific structural alterations on light microscopy. Ultrastructural examination of the ante mortem quadriceps biopsy showed several classical cap lesions, and a few of these contained small nemaline rods. The post mortem quadriceps sample showed nemaline rods in several fibres. Both samples showed mitochondrial paracrystalline inclusions in a few interstitial capillary endothelial cells. Ultrastructural findings were key in directing molecular genetic testing. A next generation sequencing panel for 35 congenital myopathy genes identified a de novo ACTA1 c.739G>C p.(Gly247Arg) variant previously reported in the literature in a patient with severe nemaline myopathy, affecting a highly conserved amino acid, and predicted to affect actin function with In Silico analysis. Our case of ACTA1-related cap-rod myopathy is the most severe presentation of a CM with caps or cap-rods described till date. The case further cements the notion of caps and rods being part of the 'nemaline spectrum' and highlights the remarkable heterogeneity of lesions within the same muscle or same group of muscles.

CONGENITAL MYOPATHIES: GENERAL AND RYR1: P.48Hypercontractile congenital muscle stiffness

Tropomyosin 3 encoded by the TPM3 gene is a member of the acting binding tropomyosin family, a component of the sarcomeric thin filaments troponin/ tropomyosin complex that is essential in muscle contraction by regulating the calcium dependent binding of the myosin head to the actin filament. Mutations in TPM3 cause a clinical and histopathological heterogeneous group of neuromuscular disorders characterized by congenital hypotonia and weakness that includes cap myopathy, congenital fiber type disproportion and nemaline myopathy. Recently, a new phenotype characterized by hypercontractile stats and stiffness was described, most probably due to mutations that increase calcium sensitivity of the troponin-tropomyosin complex, resulting in excessively sensitized excitation-contraction coupling of the contractile apparatus. This study presents the case of a one-year-old female, daughter of non-consanguineous parents, of an uneventful gestation, was born full term with Apgar 3/8 and presented generalized hypertonia and hip dysplasia from the first month of life. She had no cephalic support and was not able to sit without support because of the stiffness. Apparently, she has normal cognition. She has a short neck, hypertrophy of the cervical, paravertebral and proximal muscles of the upper limbs and a bell-shaped chest. During the neurological exam, she was able to move against gravity with all four limbs, but the strength scale evaluation was impaired due to by the stiffness. Deeper tendon reflexes, sensation and cranial nerves were normal. The first diagnosis was Schwartz Jampel syndrome; however, myotonic discharges were not detected in the electromyography exam. Computed tomography of the brain, pelvic girdle radiography, serum CK level, and mucopolysaccharidosis screen were normal. Echocardiogram showed patent foramen oval with left-right flow. Ultrasound of the hip joint showed dysplasia on the left side. Clinical, phenotypic and pathophysiological spectrum of TPM3 mutations are broader than once thought, and now joins congenital hypercontractile and stiffness phenotype.

CONGENITAL MYOPATHIES: NEMALINE AND TITINOPATHIES: P.235Recessive congenital fiber type disproportion caused by TPM3 mutation

Mutations in TPM3 gene represent a common cause of congenital fiber type disproportion (CFTD), but can also lead to other phenotypes like nemaline myopathy, cap myopathy, and congenital muscle stiffness. The clinical severity is also highly variable and genotype/phenotype correlations have been inconsistent. The majority of previously reported TPM3-associated diseases are dominantly acting, either inherited or de novo. Recessive cases are very rare and previously related to severe forms of recessive nemaline myopathy and one severely affected patient with CFTD. Here we present an autosomal recessive CFTD case harboring a novel homozygous mutation in TPM3 presenting with a mild proximal phenotype. Case Report: a 14-year-old female, only child of consanguineous parents. She was born hypotonic followed by delayed acquisition of motor milestones. Head support was achieved at the age of 6 months and gait at 30 months. At the time of the evaluation, she could walk for more than 1000 meters, climb stairs without support, but could never jump. The physical exam showed facial weakness, mild ptosis but no ophtalmoparesis and mild thoracic deformity. She presented a mild proximal cervical weakness combined with a moderate involvement of cervical flexors and no distal weakness. Muscle biopsy of the biceps brachii performed at the age of 4 years showed fiber type disproportion with type 1 predominance and smallness without other structural features like rods, nuclear centralization or cores. Whole exome sequencing revealed a novel homozygous missense variant (p.R179G) in exon 5 of TPM3 gene. The variant is not present in the population databases (EXAC, GNoMED, EVS), it is located in a very conserved residue and is predicted to be damaging according MetaSVM and MetaLR. Herein we presented a novel homozygous variant in TPM3 gene causing a mild CFTD expanding the phenotype-genotype correlations related to this gene and also adding a new variant associated with muscle disease.

Smad4 SUMOylation is essential for memory formation through upregulation of the skeletal myopathy gene TPM2

BackgroundSmad4 is a critical effector of TGF-β signaling that regulates a variety of cellular functions. However, its role in the brain has rarely been studied. Here, we examined the molecular mechanisms underlying the post-translational regulation of Smad4 function by SUMOylation, and its role in spatial memory formation.ResultsIn the hippocampus, Smad4 is SUMOylated by the E3 ligase PIAS1 at Lys-113 and Lys-159. Both spatial training and NMDA injection enhanced Smad4 SUMOylation. Inhibition of Smad4 SUMOylation impaired spatial learning and memory in rats by downregulating TPM2, a gene associated with skeletal myopathies. Similarly, knockdown of TPM2 expression impaired spatial learning and memory, while TPM2 mRNA and protein expression increased after spatial training. Among the TPM2 mutations associated with skeletal myopathies, the TPM2E122K mutation was found to reduce TPM2 expression and impair spatial learning and memory in rats.ConclusionsWe have identified a novel role of Smad4 SUMOylation and TPM2 in learning and memory formation. These results suggest that patients with skeletal myopathies who carry the TPM2E122K mutation may also have deficits in learning and memory functions.

Quaking RNA-Binding Proteins Control Early Myofibril Formation by Modulating Tropomyosin

Skeletal muscle contraction is mediated by myofibrils, complex multi-molecular scaffolds structured into repeated units, the sarcomeres. Myofibril structure and function have been extensively studied, but the molecular processes regulating its formation within the differentiating muscle cell remain largely unknown. Here we show in zebrafish that genetic interference with the Quaking RNA-binding proteins disrupts the initial steps of myofibril assembly without affecting early muscle differentiation. Using RNA sequencing, we demonstrate that Quaking is required for accumulation of the muscle-specific tropomyosin-3 transcript, tpm3.12. Further functional analyses reveal that Tpm3.12 mediates Quaking control of myofibril formation. Moreover, we identified a Quaking-binding site in the 3' UTR of tpm3.12 transcript, which is required invivo for tpm3.12 accumulation and myofibril formation. Our work uncovers a Quaking/Tpm3 pathway controlling de novo myofibril assembly. This unexpected developmental role for Tpm3 could be at the origin of muscle defects observed in human congenital myopathies associated with tpm3 mutation.

Mutation‐specific effects on thin filament length in thin filament myopathy

Thin filament myopathies are among the most common nondystrophic congenital muscular disorders, and are caused by mutations in genes encoding proteins that are associated with the skeletal muscle thin filament. Mechanisms underlying muscle weakness are poorly understood, but might involve the length of the thin filament, an important determinant of force generation. We investigated the sarcomere length-dependence of force, a functional assay that provides insights into the contractile strength of muscle fibers as well as the length of the thin filaments, in muscle fibers from 51 patients with thin filament myopathy caused by mutations in NEB, ACTA1, TPM2, TPM3, TNNT1, KBTBD13, KLHL40, and KLHL41. Lower force generation was observed in muscle fibers from patients of all genotypes. In a subset of patients who harbor mutations in NEB and ACTA1, the lower force was associated with downward shifted force-sarcomere length relations, indicative of shorter thin filaments. Confocal microscopy confirmed shorter thin filaments in muscle fibers of these patients. A conditional Neb knockout mouse model, which recapitulates thin filament myopathy, revealed a compensatory mechanism; the lower force generation that was associated with shorter thin filaments was compensated for by increasing the number of sarcomeres in series. This allowed muscle fibers to operate at a shorter sarcomere length and maintain optimal thin-thick filament overlap. These findings might provide a novel direction for the development of therapeutic strategies for thin filament myopathy patients with shortened thin filament lengths. Ann Neurol 2016;79:959-969.

New Mutations in NEB Gene Discovered by Targeted Next-Generation Sequencing in Nemaline Myopathy Italian Patients.

Nemaline myopathy represents a group of clinically and genetically heterogeneous neuromuscular disorders. Different clinical-genetic entities have been characterized in the last few years, with implications for diagnostics and genetic counseling. Fifty percent of nemaline myopathy forms are due to NEB mutations, but genetic analysis of this large and complex gene by Sanger sequencing is time consuming and expensive. We selected 10 Italian patients with clinical and biopsy features suggestive for nemaline myopathy and negative for ACTA1, TPM2 and TPM3 mutations. We applied a targeted next-generation sequencing strategy designed to analyse NEB coding regions, the relative full introns and the promoter. We also evaluated copy number variations (by CGH array) and transcriptional changes by RNA Sanger sequencing, whenever possible. This combined strategy revealed 11 likely pathogenic variants in 8 of 10 patients. The molecular diagnosis was fully achieved in 3 of 8 patients, while only one heterozygous mutation was observed in 5 subjects. This approach revealed to be a fast and cost-effective way to analyse the large NEB gene in a small group of patients and might be promising for the detection of pathological variants of other genes featuring large coding regions and lacking mutational hotspots.