The human genome contains repetitive regions, such as segmental duplications, known to be prone to copy number variation. Segmental duplications are highly identical and homologous sequences, posing ...a specific challenge for most mutation detection methods. The giant nebulin gene is expressed in skeletal muscle. It harbors a large segmental duplication region composed of eight exons repeated three times, the so-called triplicate region. Mutations in nebulin are known to cause nemaline myopathy and other congenital myopathies. Using our custom targeted Comparative Genomic Hybridization arrays, we have previously shown that copy number variations in the nebulin triplicate region are pathogenic when the copy number of the segmental duplication block deviates two or more copies from the normal number, which is three per allele. To complement our Comparative Genomic Hybridization arrays, we have established a custom Droplet Digital PCR method for the detection of copy number variations within the nebulin triplicate region. The custom Droplet Digital PCR assays allow sensitive, rapid, high-throughput, and cost-effective detection of copy number variations within this region and is ready for implementation a screening method for disease-causing copy number variations of the nebulin triplicate region. We suggest that Droplet Digital PCR may also be used in the study and diagnostics of other segmental duplication regions of the genome.
ABSTRACT
Introduction: Nebulin is a giant actin‐binding protein in the thin filament of the skeletal muscle sarcomere. Studies of nebulin interactions are limited by the size, complexity, and poor ...solubility of the protein. We divided the nebulin super‐repeat region into a super‐repeat panel, and studied nebulin/actin interactions. Methods: Actin binding was studied using a co‐sedimentation assay with filamentous actin and 26 different nebulin super‐repeats. Results: The panel revealed notable differences in actin binding between the super‐repeats. Both ends of the super‐repeat region bound actin significantly more strongly, whereas the central part of the protein bound actin weakly. Thus, the binding between nebulin and actin formed a location‐dependent pattern of strong vs. weak binding. Discussion: The nebulin super‐repeat panel allowed us to study the actin binding of each super‐repeat individually. The panel will be a powerful tool in elucidating nebulin function in health and disease. Muscle Nerve 59:116–121, 2019
Nemaline myopathy (NM) and related disorders (NMr) form a heterogenous group of ultra-rare (1:50,000 live births or less) congenital muscle disorders. To elucidate the self-reported physical, ...psychological, and social functioning in the daily lives of adult persons with congenital muscle disorders, we designed a survey using items primarily from the Patient Reported Outcomes Measurement Information System, PROMISR, and conducted a pilot study in patients with NM and NMr in Finland. The items were linked to International Classification of Functioning, Disability and Health (ICF) categories. In total, 20 (62.5%) out of 32 invited persons resident in Finland participated in the study; 12 had NM and 8 NMr, 15 were women and 5 men aged 19-75 years. Sixteen (80%) were ambulatory and 4 (20%) NM patients used wheelchairs. The results from the PROMIS measuring system and ICF categories both indicated that non-ambulatory patients of this study faced more challenges in all areas of functioning than ambulatory ones, but the differences were smaller in the domains measuring psychological and social functioning than in physical functioning. In addition, the COVID-19 pandemic adversely affected the functioning of non-ambulatory patients more than that of ambulatory patients. The interindividual differences were, however, noticeable. To our knowledge, this pilot study is the first comprehensive survey-based study of the physical, psychological, and social functioning of adult persons with nemaline myopathy or related disorders. The results indicate vulnerability of non-ambulatory patients being at higher risk to a decrease in general functioning during global or national exceptional periods. The responses also gave directions for modifying and improving the survey for future studies.
Objective
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.
Methods
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.
Results
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.
Interpretation
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
ABSTRACT
A mutation update on the nebulin gene (NEB) is necessary because of recent developments in analysis methodology, the identification of increasing numbers and novel types of variants, and a ...widening in the spectrum of clinical and histological phenotypes associated with this gigantic, 183 exons containing gene. Recessive pathogenic variants in NEB are the major cause of nemaline myopathy (NM), one of the most common congenital myopathies. Moreover, pathogenic NEB variants have been identified in core‐rod myopathy and in distal myopathies. In this update, we present the disease‐causing variants in NEB in 159 families, 143 families with NM, and 16 families with NM‐related myopathies. Eighty‐eight families are presented here for the first time. We summarize 86 previously published and 126 unpublished variants identified in NEB. Furthermore, we have analyzed the NEB variants deposited in the Exome Variant Server (http://evs.gs.washington.edu/EVS/), identifying that pathogenic variants are a minor fraction of all coding variants (∼7%). This indicates that nebulin tolerates substantial changes in its amino acid sequence, providing an explanation as to why variants in such a large gene result in relatively rare disorders. Lastly, we discuss the difficulties of drawing reliable genotype–phenotype correlations in NEB‐associated disease.
Variants in the nebulin gene are the most common cause of autosomal recessive nemaline myopathy, but the clinical and histological spectrum is a continuum ranging from severe to mild forms of congenital myopathies. The spectrum of pathogenic variants range from point mutations to large deletions and duplications covering large parts of the gene. Amino acid changes are very common in nebulin, but only few of them are disease‐causing. Detailed genotype‐phenotype correlations are difficult to discern.
ABSTRACT
Mutations affecting skeletal muscle isoforms of the tropomyosin genes may cause nemaline myopathy, cap myopathy, core‐rod myopathy, congenital fiber‐type disproportion, distal ...arthrogryposes, and Escobar syndrome. We correlate the clinical picture of these diseases with novel (19) and previously reported (31) mutations of the TPM2 and TPM3 genes. Included are altogether 93 families: 53 with TPM2 mutations and 40 with TPM3 mutations. Thirty distinct pathogenic variants of TPM2 and 20 of TPM3 have been published or listed in the Leiden Open Variant Database (http://www.dmd.nl/). Most are heterozygous changes associated with autosomal‐dominant disease. Patients with TPM2 mutations tended to present with milder symptoms than those with TPM3 mutations, DA being present only in the TPM2 group. Previous studies have shown that five of the mutations in TPM2 and one in TPM3 cause increased Ca2+ sensitivity resulting in a hypercontractile molecular phenotype. Patients with hypercontractile phenotype more often had contractures of the limb joints (18/19) and jaw (6/19) than those with nonhypercontractile ones (2/22 and 1/22), whereas patients with the non‐hypercontractile molecular phenotype more often (19/22) had axial contractures than the hypercontractile group (7/19). Our in silico predictions show that most mutations affect tropomyosin–actin association or tropomyosin head‐to‐tail binding.
We present mutation update and genotype‐phenotype correlations for novel (16) and previously reported (31) mutations of the TPM2 and TPM3 genes causing nemaline myopathy, cap myopathy, core‐rod myopathy, congenital fibre‐type disproportion, distal arthrogryposes and Escobar syndrome. Included are 93 families: 53 with TPM2 mutations and 40 with TPM3 mutations. Six mutations cause increased Ca2+ sensitivity resulting in hypercontractile phenotypes. Our in silico predictions show that most mutations affect tropomyosin‐actin association or tropomyosin head‐to‐tail binding.
Intragenic segmental duplication regions are potential hotspots for recurrent copy number variation and possible pathogenic aberrations. Two large sarcomeric genes, nebulin and titin, both contain ...such segmental duplication regions. Using our custom Comparative Genomic Hybridisation array, we have previously shown that a gain or loss of more than one copy of the repeated block of the nebulin triplicate region constitutes a recessive pathogenic mutation. Using targeted array-CGH, similar copy number variants can be detected in the segmental duplication region of titin. Due to the limitations of the array-CGH methodology and the repetitiveness of the region, the exact copy numbers of the blocks could not be determined. Therefore, we developed complementary custom Droplet Digital PCR assays for the titin segmental duplication region to confirm true variation. Our combined methods show that the titin segmental duplication region is subject to recurrent copy number variation. Gains and losses were detected in samples from healthy individuals as well as in samples from patients with different muscle disorders. The copy number variation observed in our cohort is likely benign, but pathogenic copy number variants in the segmental duplication region of titin cannot be excluded. Further investigations are needed, however, this region should no longer be neglected in genetic analyses.
Nebulin is a giant protein expressed at high levels in skeletal muscle. Mutations in the nebulin gene (NEB) lead to muscle weakness and various congenital myopathies. Despite increasing clinical and ...scientific interest, the pathogenesis of weakness remains unknown. The present study, therefore, aims at unraveling the underlying molecular mechanisms. Hence, we recorded and analyzed the mechanics as well as the X‐ray diffraction patterns of human membrane‐perme‐abilized single muscle fibers expressing nebulin mutations. Results demonstrated that, during contraction, the cycling rate of myosin heads attaching to actin is dramatically perturbed, causing a reduction in the fraction of myosin‐actin interactions in the strong binding state. This phenomenon prevents complete thin‐filament activation, more especially proper and full tropomyosin movement, further limiting additional binding of myosin cross‐bridges. At the cell level, this reduces the force‐generating capacity and, overall, provokes muscle weakness. To reverse such a negative cascade of events, future potential therapeutic interventions should, therefore, focus on the triggering component, the altered myosin cross‐bridge cycling kinetics.—Ochala, J., Lehtokari, V.‐L., Iwamoto, H., Li, M., Feng, H.‐Z., Jin, J. P., Yagi, N., Wallgren‐Pettersson, C. Pénisson‐Besnier, I., Larsson, L. Disrupted myosin cross‐bridge cycling kinetics triggers muscle weakness in nebulin‐related myopathy. FASEB J. 25, 1903‐1913 (2011). www.fasebj.org
•Novel clinical finding of muscle hypertonia in tropomyosin-caused congenital myopathy.•Unusual symptom of trismus.•Unspecific histological findings.
Patients with myopathies caused by pathogenic ...variants in tropomyosin genes TPM2 and TPM3 usually have muscle hypotonia and weakness, their muscle biopsies often showing fibre size disproportion and nemaline bodies. Here, we describe a series of patients with hypercontractile molecular phenotypes, high muscle tone, and mostly non-specific myopathic biopsy findings without nemaline bodies. Three of the patients had trismus, whilst in one patient, the distal joints of her fingers flexed on extension of the wrists. In one biopsy from a patient with a rare TPM3 pathogenic variant, cores and minicores were observed, an unusual finding in TPM3-caused myopathy. The variants alter conserved contact sites between tropomyosin and actin.
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