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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.

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.

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 Hybridization 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 variants, which is surprisingly common. 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.

Nemaline myopathy (NM), a structural congenital myopathy, presents a significant clinical and genetic heterogeneity. Here, we compiled molecular and clinical data of 30 Brazilian patients from 25 unrelated families. Next-generation sequencing was able to genetically classify all patients: sixteen families (64%) with mutation in NEB, five (20%) in ACTA1, two (8%) in KLHL40, and one in TPM2 (4%) and TPM3 (4%). In the NEB-related families, 25 different variants, 11 of them novel, were identified; splice site (10/25) and frame shift (9/25) mutations were the most common. Mutation c.24579 G>C was recurrent in three unrelated patients from the same region, suggesting a common ancestor. Clinically, the “typical” form was the more frequent and caused by mutations in the different NM genes. Phenotypic heterogeneity was observed among patients with mutations in the same gene. Respiratory involvement was very common and often out of proportion with limb weakness. Muscle MRI patterns showed variability within the forms and genes, which was related to the severity of the weakness. Considering the high frequency of NEB mutations and the complexity of this gene, NGS tools should be combined with CNV identification, especially in patients with a likely non-identified second mutation.

We report the first mosaic mutation, a deletion of exons 11-107, identified in the nebulin gene in a Finnish patient presenting with a predominantly distal congenital myopathy and asymmetric muscle weakness. The female patient is ambulant and currently 26 years old. Muscle biopsies showed myopathic features with type 1 fibre predominance, strikingly hypotrophic type 2 fibres and central nuclei, but no nemaline bodies. The deletion was detected in a copy number variation analysis based on next-generation sequencing data. The parents of the patient did not carry the deletion. Mosaicism was detected using a custom, targeted comparative genomic hybridisation array. Expression of the truncated allele, less than half the size of full-length nebulin, was confirmed by Western blotting. The clinical and histological picture resembled that of a family with a slightly smaller deletion, and that in patients with recessively inherited distal forms of nebulin-caused myopathy. Asymmetry, however, was a novel feature. (c) 2021 Elsevier B.V. All rights reserved.

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.

We present here a Finnish nemaline myopathy family with a dominant mutation in the skeletal muscle alpha-actin gene, p.(Glu85Lys), segregating in three generations. The index patient, a 5-year-old boy, had the typical form of nemaline myopathy with congenital muscle weakness and motor milestones delayed but reached, while his mother never had sought medical attention for her very mild muscle weakness, and his maternal grandmother had been misdiagnosed as having myotonic dystrophy. This illustrates the clinical variability in nemaline myopathy. (C) 2017 Elsevier B.V. All rights reserved.

Objective To examine the occurrence, course, and clinical picture of the DSM-5 residual categories ‘Other Specified Feeding or Eating Disorder’ (OSFED) and ‘Unspecified Feeding or Eating Disorder’ (UFED), to describe potential subtypes, and to evaluate whether the subdivision of the residual category appears meaningful. Method We screened women from the 1975–79 birth cohorts of Finnish twins (N = 2825) for lifetime eating disorders using questionnaires and the SCID interview. This analysis characterizes women who reported clinically significant eating disorder symptoms but did not fulfill diagnostic criteria for DSM-5 anorexia nervosa (AN), bulimia nervosa (BN), or binge eating disorder (BED). Results Thirty-eight women (21% of those with an eating disorder) fell in the residual OSFED/UFED category. A third of them (N = 14) fulfilled OSFED criteria, whereas two thirds (N = 24) fell in the UFED category. The lifetime prevalence of OSFED/UFED was 1.5% (95% CI 1.1–2.0%), less than half of the prevalence of DSM-IV eating disorder not otherwise specified (EDNOS). The mean age of onset of OSFED/UFED was 18 years, median duration of symptoms was two years, and the 5-year probability of recovery was 60%. Over a third of women with OSFED/UFED suffered from comorbid psychiatric disorders. Both residual categories were clinically heterogeneous and included atypical forms of AN, BN, and BED. Conclusions Applying the DSM-5 criteria in a community sample of young women more than halved the occurrence of residual eating disorder diagnoses, but resulted in two instead of one clinically heterogeneous residual categories. Nevertheless, residual eating disorders were associated with considerable clinical severity. © 2016 Wiley Periodicals, Inc. (Int J Eat Disord 2016; 49:1010–1017)

Nemaline myopathy and congenital fiber type disproportion are among the most common nondystrophic congenital muscular disorders.1 Genes that are implicated in these myopathies encode proteins that are either components of the skeletal muscle thin filament, including nebulin (NEB), skeletal muscle alpha-actin1 (ACTA1), beta-tropomyosin 2 (TPM2), alpha-tropomyosin 3 (TPM3), troponin T type 1 (TNNT1), cofilin-2 (CFL2), and leiomodin-3 (LMOD3), or are thought to contribute to stability or turnover of thin filament proteins, such as kelch repeat and BTB (POZ) domain containing 13 (KBTBD13), and kelchlike family members 40 (KLHL40) and 41 (KLHL41).2–6 Hence, muscle diseases caused by mutations in these genes are here referred to as thin filament myopathies. For a schematic of the thin filament and its associated proteins, see Figure 1. Patients with thin filament myopathy suffer from muscle weakness, but the underlying mechanisms are poorly understood. FIGURE 1 Schematic of the skeletal muscle thin filament. The thin filament is an essential structure in the sarcomere, the smallest contractile unit in skeletal muscle. The actin-based backbone of the thin filament is decorated with proteins that are involved ... The thin filament is a major constituent of the sarcomere, the smallest contractile unit in muscle, and is essential for force generation; its length determines the overlap between the thin and thick filament, and thereby the number of force-generating interactions that can be formed between actin and myosin. In healthy human muscle, the length of the thick filament is 1.6μm and that of the thin filament is regulated at 1.1 to 1.3μm.7 Accordingly, force depends on sarcomere length, with increasing force as the overlap between thick and thin filaments increases (up to a sarcomere length of ~2.6μm; ie, the ascending limb of the force–sarcomere length relation), and decreasing force at longer sarcomere lengths as the overlap between thick and thin filaments decreases (ie, the descending limb; Fig 2A8,9). Hence, appropriate length of the thin filament is important for muscle fiber strength; a shorter length causes lower force generation by shifting the descending limb of the force–sarcomere length relation downward. FIGURE 2 The force–sarcomere length relation in muscle fibers from thin filament myopathy patients and control (CTRL) subjects. (A) The shape of the force–sarcomere length relation is determined by the amount of overlap between the thick and thin ... Whether mutations in genes implicated in thin filament myopathy contribute to force loss by affecting thin filament length is unclear. Mouse models with mutations in Neb exhibit a shorter thin filament length associated with lower force generation that becomes more prominent as sarcomere length increases.10–12 Preliminary studies on a small number of human biopsies were largely in agreement with this observation.13,14 Whether these findings translate to a large group of patients, and whether shorter thin filament length is a general mechanism underlying force loss in thin filament myopathies with a variety of different gene defects, is unknown. Therefore, we studied muscle fibers from 51 patients with thin filament myopathy caused by mutations in NEB, ACTA1, TPM2, TPM3, TNNT1, KBTBD13, KLHL40, and KLHL41. In these fibers, we determined the sarcomere length-dependence of force, a functional assay that provides insight into the contractile strength of muscle fibers as well as the length of the thin filaments. In a novel, conditional Neb knockout mouse model that recapitulates thin filament myopathy—including shorter thin filament length—we studied whether muscle possesses mechanisms that compensate for shorter thin filament length, and we specifically focused on the addition of sarcomeres in series.

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.Actin binding was studied using a co-sedimentation assay with filamentous actin and 26 different nebulin super-repeats.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.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.

Background and Objectives: Nemaline myopathy may be caused by pathogenic variants in the TPM3 gene and is then called NEM1. All previously identified disease-causing variants are point mutations including missense, nonsense and splice-site variants. The aim of the study was to identify the disease-causing gene in this patient and verify the NM diagnosis. Methods: Mutation analysis methods include our self-designed nemaline myopathy array, The Nemaline Myopathy Comparative Genomic Hybridisation Array (NM-CGH array), whole-genome array-CGH, dHPLC, Sanger sequencing and whole-exome sequencing. The diagnostic muscle biopsy was investigated further by routine histopathological methods. Results: We present here the first large (17–21 kb) aberration in the α-tropomyosinslow gene (TPM3), identified using the NM-CGH array. This homozygous deletion removes the exons 1a and 2b as well as the promoter of the TPM3 isoform encoding Tpm3.12st. The severe phenotype included paucity of movement, proximal and axial weakness and feeding difficulties requiring nasogastric tube feeding. The infant died at the age of 17.5 months. Muscle biopsy showed variation in fibre size and rods in a population of hypotrophic muscle fibres expressing slow myosin, often with internal nuclei, and abnormal immunolabelling revealing many hybrid fibres. Conclusions: This is the only copy number variation we have identified in any NM gene other than nebulin (NEB), suggesting that large deletions or duplications in these genes are very rare, yet possible, causes of NM.

International audience; Interpretation of next-generation sequencing constitutes the main limitation of molecular diagnostics. In diagnosing myopathies and muscular dystrophies, another issue is efficiency in predicting the pathogenicity of variants identified in large genes, especially TTN; current in silico prediction tools show limitations in predicting and ranking the numerous variants of such genes. We propose a variant-prioritization tool, the MoBiDiC prioritization algorithm (MPA). MPA is based on curated interpretation of data on previously reported variants, biological assumptions, and splice and missense predictors, and is used to prioritize all types of single-nucleotide variants. MPA was validated by comparing its sensitivity and specificity to those of dbNSFP database prediction tools, using a data set composed of DYSF, DMD, LMNA, NEB, and TTN variants extracted from expert-reviewed and ExAC databases. MPA obtained the best annotation rates for missense and splice variants. As MPA aggregates the results from several predictors, individual predictor errors are counterweighted, improving the sensitivity and specificity of missense and splice variant predictions. We propose a sequential use of MPA, beginning with the selection of variants with higher scores and followed by, in the absence of candidate pathologic variants, consideration of variants with lower scores. We provide scripts and documentation for free academic use and a validated annotation pipeline scaled for panel and exome sequencing to prioritize single-nucleotide variants from a VCF file.

Background: Our previous array, the Comparative Genomic Hybridisation design (CGH-array) for nemaline myopathy (NM), named the NM-CGH array, revealed pathogenic copy number variation (CNV) in the genes for nebulin (NEB) and tropomyosin 3 (TPM3), as well as recurrent CNVs in the segmental duplication (SD), i.e. triplicate, region of NEB (TRI, exons 82-89, 90-97, 98-105). In the light of this knowledge, we have designed and validated an extended CGH array, which includes a selection of 187 genes known to cause neuromuscular disorders (NMDs). Objective: Our aim was to develop a reliable method for CNV detection in genes related to neuromuscular disorders for routine mutation detection and analysis, as a much-needed complement to sequencing methods. Methods: We have developed a novel custom-made 4×180 k CGH array for the diagnostics of NMDs. It includes the same tiled ultra-high density coverage of the 12 known or putative NM genes as our 8×60 k NM-CGH-array but also comprises a selection of 175 additional genes associated with NMDs, including titin (TTN), at a high to very high coverage. The genes were divided into three coverage groups according to known and potential pathogenicity in neuromuscular disorders. Results: The array detected known and putative CNVs in all three gene coverage groups, including the repetitive regions of NEB and TTN. Conclusions: The targeted neuromuscular disorder 4×180 k array-CGH (NMD-CGH-array v1.0) design allows CNV detection for a broader spectrum of neuromuscular disorders at a high resolution. © 2018 - IOS Press and the authors. All rights reserved.

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.

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 Ca(2+) 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.

Mutations in the TPM2 gene, which encodes β-tropomyosin, are an established cause of several congenital skeletal myopathies and distal arthrogryposis. We have identified a TPM2 mutation, p.K7del, in five unrelated families with nemaline myopathy and a consistent distinctive clinical phenotype. Patients develop large joint contractures during childhood, followed by slowly progressive skeletal muscle weakness during adulthood. The TPM2 p.K7del mutation results in the loss of a highly conserved lysine residue near the N-terminus of β-tropomyosin, which is predicted to disrupt head-to-tail polymerization of tropomyosin. Recombinant K7del-β-tropomyosin incorporates poorly into sarcomeres in C2C12 myotubes and has a reduced affinity for actin. Two-dimensional gel electrophoresis of patient muscle and primary patient cultured myotubes showed that mutant protein is expressed but incorporates poorly into sarcomeres and likely accumulates in nemaline rods. In vitro studies using recombinant K7del-β-tropomyosin and force measurements from single dissected patient myofibres showed increased myofilament calcium sensitivity. Together these data indicate that p.K7del is a common recurrent TPM2 mutation associated with mild nemaline myopathy. The p.K7del mutation likely disrupts head-to-tail polymerization of tropomyosin, which impairs incorporation into sarcomeres and also affects the equilibrium of the troponin/tropomyosin-dependent calcium switch of muscle. Joint contractures may stem from chronic muscle hypercontraction due to increased myofibrillar calcium sensitivity while declining strength in adulthood likely arises from other mechanisms, such as myofibre decompensation and fatty infiltration. These results suggest that patients may benefit from therapies that reduce skeletal muscle calcium sensitivity, and we highlight late muscle decompensation as an important cause of morbidity. * Abbreviations : CCD : charge-coupled device CD : circular dichroism DTT : dithiothreitol EGFP : enhanced green fluorescent protein EGTA : ethylene glycol-bis tetraacetic acid IPG : immobilised pH gradient K7del-βTm : K7del-β-tropomyosin recombinant protein MES : morpholinoethanesulfonic acid NADH-TR : nicotinamide adenine dinucleotide hydroxyl tetrazolium reductase NHLBI : The National Heart Lung and Blood Institute SAXS : small-angle X-ray scattering WT-βTm : wild-type β-tropomyosin

Objective To increase awareness to the possibility of nemaline myopathy (NM) when abnormal prenatal ultrasound findings appear together with a carrier state for the common exon 55 deletion in the nebulin gene (NEB) of an Ashkenazi Jewish parent. Methods We describe four unrelated pregnancies with abnormal prenatal ultrasound findings resulting in the birth of newborns with NM, where one or both parents were of Ashkenazi Jewish origin. Data was collected retrospectively from the patients' medical files. Molecular analysis of NEB was performed on the DNA from the patients and parents. Results Prenatal ultrasound findings included polyhydramnios, decreased fetal movements, club feet, and arthrogryposis. A biopsy from two of the newborns was consistent with NM. In all of the newborns, the common NEB exon 55 deletion was detected in the heterozygote state and in three of them, a second novel mutation was found. Conclusions Ultrasonographic findings suggestive of a myopathy and a carrier state for the NEB exon 55 deletion in one of the parents should trigger a thorough investigation for NM. The extreme size of NEB imposes great difficulties when searching for a second mutation, especially under the time constraints of an ongoing pregnancy. © 2012 John Wiley & Sons, Ltd.

Mutations in the nebulin gene are the main cause of autosomal recessive nemaline myopathy, with clinical presentations ranging from mild to severe disease. We have previously reported a nonspecific distal myopathy caused by homozygous missense mutations in the nebulin gene in six Finnish patients from four different families. Here we describe three non-Finnish patients in two unrelated families with distal nemaline myopathy caused by four different compound heterozygous nebulin mutations, only one of which is a missense mutation. One of the mutations has previously been identified in one family with the severe form of nemaline myopathy. We conclude that nemaline myopathy and distal myopathy caused by nebulin mutations form a clinical and histological continuum. Nemaline myopathy should be considered as a differential diagnosis in patients presenting with an early-onset predominantly distal myopathy.

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-permeabilized 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.

‘‘Cap myopathy’’ or ‘‘cap disease’’ is a congenital myopathy characterised by cap-like structures at the periphery of muscle fibres, consisting of disarranged thin filaments with enlarged Z discs. Here we report a deletion in the b-tropomyosin (TPM2) gene causing cap disease in a 36-year-old male patient with congenital muscle weakness, myopathic facies and respiratory insufficiency. The mutation identified in this patient is an in-frame deletion (c.415_417delGAG) of one codon in exon 4 of TPM2 removing a single glutamate residue (p.Glu139del) from the b-tropomyosin protein. This is expected to disrupt the seven-amino acid repeat essential for making a coiled coil, and thus to impair tropomyosin–actin interaction. Missense mutations in TPM2 have previously been found to cause rare cases of nemaline myopathy and distal arthrogryposis. This mutation is one not previously described and the first genetic cause identified for cap disease. � 2007 Elsevier B.V. All rights reserved.

We describe a novel, recessively inherited distal myopathy caused by homozygous missense mutations in the nebulin gene (NEB), in which other combinations of mutations are known to cause nemaline (rod) myopathy (NM). Two different missense mutations were identified in homozygous form in seven Finnish patients from four unrelated families with childhood or adult-onset foot drop. Both mutations, when combined in compound heterozygous form with more disruptive mutations in NEB, are known to cause NM. Hitherto, no patients with NM have been found to have two missense mutations in NEB. Muscle weakness predominantly affected ankle dorsiflexors, finger extensors and neck flexors, a distribution different both from the patterns of weakness seen in NM caused by NEB mutations, and those of the known recessively inherited distal myopathies. Singleton cases need to be distinguished from the Laing type of distal myopathy. Histologically, this myopathy differs from NM in that nemaline bodies were not detectable with routine light microscopy, and they were inconspicuous or absent even with electron microscopy. Rimmed vacuoles, commonly seen in other distal myopathies, were not a feature. We conclude that homozygous missense mutations in NEB cause a novel distal myopathy, predominantly involving lower leg extensor muscles, finger extensors and neck flexors.

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.

Background Congenital myopathies (CM) are a group of rare inherited muscle disorders characterized by particular histopathological alterations on muscle biopsy. Core-rod myopathy is a CM presenting with cores and rods as distinctive muscle morphological features. Methods/results: We describe 3 young patients presenting congenital core-rod myopathy with bilateral foot-drop associated with autosomal recessive nebulin gene (NEB) mutations detected by exome sequencing. Conclusions This report illustrates that core-rod congenital myopathy with foot-drop is frequently associated with NEB gene mutations and should be considered in the differential diagnosis of early onset distal myopathies.

The giant nebulin protein is a fundamental structural component of the thin filaments of the striated muscle sarcomere. Nebulin binds to actin and the size of nebulin correlates with actin filament length, suggesting that nebulin may determine the length of the thin filaments during myofibrillogenesis. We have previously described the genomic organization of the 3' end of the nebulin gene (NEB), and identified 18 different NEB mutations in patients with autosomal recessive nemaline myopathy. Here we present the genomic organization of the entire nebulin gene, and the identification of numerous alternatively spliced mRNAs. The gene comprises 183 exons spanning 249 kb of the genomic sequence. The translation initiation codon is in exon 3, and the stop codon and the 3' UTR are in exon 183. There are four regions with alternatively spliced exons, that is, exons 63-66, 82-105, 143-144 and 166-177, giving rise to a number of different transcripts. The alternatively spliced exons 143-144 give rise to two different transcripts varying between muscle types and between muscles of different developmental stages. The alternatively spliced exons 166-177 express at least 20 different transcripts in adult human tibialis anterior muscle alone. Preliminary results show several transcripts in both of the two remaining alternatively spliced regions. Extensive alternative splicing of NEB may explain why nemaline myopathy patients with homozygous truncating mutations show expression of the carboxy-terminus of the nebulin protein contrary to expectations. The use of alternative transcripts might also explain why severe phenotypes are rare among patients with two truncating mutations.

Nemaline myopathy (NM) is a genetic muscle disorder characterized by muscle dysfunction and electron-dense protein accumulations (nemaline bodies) in myofibers. Pathogenic mutations have been described in 9 genes to date, but the genetic basis remains unknown in many cases. Here, using an approach that combined whole-exome sequencing (WES) and Sanger sequencing, we identified homozygous or compound heterozygous variants in LMOD3 in 21 patients from 14 families with severe, usually lethal, NM. LMOD3 encodes leiomodin-3 (LMOD3), a 65-kDa protein expressed in skeletal and cardiac muscle. LMOD3 was expressed from early stages of muscle differentiation; localized to actin thin filaments, with enrichment near the pointed ends; and had strong actin filament-nucleating activity. Loss of LMOD3 in patient muscle resulted in shortening and disorganization of thin filaments. Knockdown of lmod3 in zebrafish replicated NM-associated functional and pathological phenotypes. Together, these findings indicate that mutations in the gene encoding LMOD3 underlie congenital myopathy and demonstrate that LMOD3 is essential for the organization of sarcomeric thin filaments in skeletal muscle.

Nemaline myopathy (NEM) is a common congenital myopathy. At the very severe end of the NEM clinical spectrum are genetically unresolved cases of autosomal-recessive fetal akinesia sequence. We studied a multinational cohort of 143 severe-NEM-affected families lacking genetic diagnosis. We performed whole-exome sequencing of six families and targeted gene sequencing of additional families. We identified 19 mutations in KLHL40 (kelch-like family member 40) in 28 apparently unrelated NEM kindreds of various ethnicities. Accounting for up to 28% of the tested individuals in the Japanese cohort, KLHL40 mutations were found to be the most common cause of this severe form of NEM. Clinical features of affected individuals were severe and distinctive and included fetal akinesia or hypokinesia and contractures, fractures, respiratory failure, and swallowing difficulties at birth. Molecular modeling suggested that the missense substitutions would destabilize the protein. Protein studies showed that KLHL40 is a striated-muscle-specific protein that is absent in KLHL40-associated NEM skeletal muscle. In zebrafish, klhl40a and klhl40b expression is largely confined to the myotome and skeletal muscle, and knockdown of these isoforms results in disruption of muscle structure and loss of movement. We identified KLHL40 mutations as a frequent cause of severe autosomal-recessive NEM and showed that it plays a key role in muscle development and function. Screening of KLHL40 should be a priority in individuals who are affected by autosomal-recessive NEM and who present with prenatal symptoms and/or contractures and in all Japanese individuals with severe NEM.

Nemaline myopathy (NM) constitutes a heterogeneous group of congenital myopathies. Mutations in the nebulin gene (NEB) are the main cause of recessively inherited NM. NEB is one of the most largest genes in human. To date, 68 NEB mutations, mainly small deletions or point mutations have been published. The only large mutation characterized is the 2.5 kb deletion of exon 55 in the Ashkenazi Jewish population. To investigate any copy number variations in this enormous gene, we designed a novel custom comparative genomic hybridization microarray, NM-CGH, targeted towards the seven known genes causative for NM. During the validation of the NM-CGH array we identified two novel deletions in two different families. The first is the largest deletion characterized in NEB to date, (∼53 kb) encompassing 24 exons. The second deletion (1 kb) covers two exons. In both families, the copy number change was the second mutation to be characterized and shown to have been inherited from one of the healthy carrier parents. In addition to these novel mutations, copy number variation was identified in four samples in three families in the triplicate region of NEB. We conclude that this method appears promising for the detection of copy number variations in NEB.

Nemaline myopathy, also called rod myopathy, is a relatively common congenital myopathy and probably second in incidence only to central core disease. The mainstay of diagnosis is histopathology, but detection of the causative mutation is mandatory for determining the mode of inheritance and for prenatal diagnosis. The authors report two siblings with nemaline myopathy caused by mutations in the nebulin gene.

To increase awareness to the possibility of nemaline myopathy (NM) when abnormal prenatal ultrasound findings appear together with a carrier state for the common exon 55 deletion in the nebulin gene (NEB) of an Ashkenazi Jewish parent.We describe four unrelated pregnancies with abnormal prenatal ultrasound findings resulting in the birth of newborns with NM, where one or both parents were of Ashkenazi Jewish origin. Data was collected retrospectively from the patients' medical files. Molecular analysis of NEB was performed on the DNA from the patients and parents.Prenatal ultrasound findings included polyhydramnios, decreased fetal movements, club feet, and arthrogryposis. A biopsy from two of the newborns was consistent with NM. In all of the newborns, the common NEB exon 55 deletion was detected in the heterozygote state and in three of them, a second novel mutation was found.Ultrasonographic findings suggestive of a myopathy and a carrier state for the NEB exon 55 deletion in one of the parents should trigger a thorough investigation for NM. The extreme size of NEB imposes great difficulties when searching for a second mutation, especially under the time constraints of an ongoing pregnancy.

A commentary on identification of the rare compound heterozygous variants in the NEB gene in a Korean family with intellectual disability, epilepsy and early-childhood-onset generalized muscle weakness

Nemaline myopathy (NM) and central core disease (CCD), 2 of the most frequent congenital myopathies, are clinically and genetically heterogeneous disorders of skeletal muscle. NM (OMIM 161800) is caused by mutations in at least 6 different genes encoding thin filament proteins of the striated muscle sarcomere: skeletal alpha-actin ( ACTA1 ), nebulin ( NEB ), beta-tropomyosin ( TPM2 ), slow alpha-tropomyosin ( TPM3 ), slow skeletal muscle troponin-T ( TNNT1 ), and cofilin-2 ( CFL2 ). CCD (OMIM 117000) is mainly caused by mutations in the gene encoding the skeletal muscle ryanodine receptor ( RYR1 ), a Ca2+ channel involved in excitation-contraction coupling. Congenital myopathies with the combination of distinct “cores” and “rods” in the same muscle fibers have been described in a small number of cases. In 2 families mutations have been identified in RYR1 1,2 and in others, linkage has been found to a locus on chromosome 15.3 Here we report a patient with recessively inherited core-rod myopathy caused by compound heterozygous mutations in NEB . This observation broadens the spectrum of genes involved in core-rod myopathies. ### Case report. The patient is a 27-year-old white man. He presented at birth with generalized hypotonia and required immediate intubation and resuscitation. Spontaneous movements were very poor and he had seizures from the first week. Restrictive respiratory insufficiency led to continuous mechanical ventilation via tracheotomy at the age of 2 years. The patient did not acquire independent walking but was able to take a few steps with orthosis at the age of 4; diffuse muscle weakness with axial predominance was observed. Electromyography showed myopathic patterns and normal motor and sensory responses. Serum creatine kinase levels were normal. The patient developed …

In 2004, Anderson et al. reported a homozygous 2502 bp deletion including exon 55 of the nebulin gene in five Ashkenazi Jewish probands with nemaline myopathy. We determined the occurrence of this deletion in a world-wide series of 355 nemaline myopathy probands with no previously known mutation in other genes and found the mutation in 14 probands, two of whom represented families previously ascertained by Anderson et al. Two of the families were not of known Ashkenazi Jewish descent but they had the haplotype known to segregate with this mutation. In all but two of eight homozygous patients, the clinical picture was more severe than in typical nemaline myopathy.

To date, six genes are known to cause nemaline (rod) myopathy (NM), a rare congenital neuromuscular disorder. In an attempt to find a seventh gene, we performed linkage and subsequent sequence analyses in 12 Turkish families with recessive NM. We found homozygosity in two of the families at 1q12-21.2, a region encompassing the gamma-tropomyosin gene (TPM3) encoding slow skeletal muscle alpha-tropomyosin, a known NM gene. Sequencing revealed homozygous deletion of the first nucleotide of the last exon, c.913delA of TPM3 in both families. The mutation removes the last nucleotide before the stop codon, causing a frameshift and readthrough across the termination signal. The encoded alphaTm(slow) protein is predicted to be 73 amino acids longer than normal, and the extension to the protein is hypothesised to be unable to form a coiled coil. The resulting tropomyosin protein may therefore be non-functional. The affected children in both families were homozygous for the mutation, while the healthy parents were mutation carriers. Both of the patients in Family 1 had the severe form of NM, and also an unusual chest deformity. The affected children in Family 2 had the intermediate form of NM. Muscle biopsies showed type 1 (slow) fibres to be markedly smaller than type 2 (fast) fibres. Previously, there had been five reports, only, of NM caused by mutations in TPM3. The mutation reported here is the first deletion to be identified in TPM3, and it is likely to be a founder mutation in the Turkish population.

Nemaline myopathy (NM) is a clinically and genetically heterogeneous disorder of skeletal muscle caused by mutations in at least five different genes encoding thin filament proteins of the striated muscle sarcomere. We have previously described 18 different mutations in the last 42 exons of the nebulin gene (NEB) in 18 families with NM. Here we report 45 novel NEB mutations detected by denaturing high-performance liquid chromatography (dHPLC) and sequence analysis of all 183 NEB exons in NM patients from 44 families. Altogether we have identified, including the deletion of exon 55 identified in the Ashkenazi Jewish population, 64 different mutations in NEB segregating with autosomal recessive NM in 55 families. The majority (55%) of the mutations in NEB are frameshift or nonsense mutations predicted to cause premature truncation of nebulin. Point mutations (25%) or deletions (3%) affecting conserved splice signals are predicted in the majority of cases to cause in-frame exon skipping, possibly leading to impaired nebulin-tropomyosin interaction along the thin filament. Patients in 18 families had one of nine missense mutations (14%) affecting conserved amino acids at or in the vicinity of actin or tropomyosin binding sites. In addition, we found the exon 55 deletion in four families. The majority of the patients (in 49/55 families) were shown to be compound heterozygous for two different mutations. The mutations were found in both constitutively and alternatively expressed exons throughout the NEB gene, and there were no obvious mutational hotspots. Patients with more severe clinical pictures tended to have mutations predicted to be more disruptive than patients with milder forms.

One of the key components in stabilizing and determining the lengths of the actin filaments of the skeletal muscle sarcomere and in the neurons of the brain is the gigantic protein nebulin. Its size varies from 600 to 900 kDa and correlates with the length of the actin filaments. A huge variation in length is produced by differential usage of the alternatively spliced exons 63–66, 143–144 and 166–177, among the 183 exons of the nebulin gene (NEB). Exons 63–66 are always expressed together as a block, while exons 143 and 144 give rise to two different transcripts varying between muscle types and in different developmental stages. Exons 166–177 are spliced independently. Over 150 recessive, mostly nemaline myopathy- (NM) causing mutations, but also mutations causing core-rod and distal myopathies have been identified to date; homozygous or both of the compound heterozygous mutations have been identified in 106 probands of the Helsinki cohort. Of these probands, 23 have a mutation in an alternatively spliced exon. Interestingly, at least twelve of them have clinical features unusual in NM, and two have core-rod myopathy. Vice versa, many probands in the Helsinki cohort with NEB mutations and unusual features such as core structures seen in their muscle biopsies have at least one of their two compound heterozygous mutations located in an alternatively spliced exon; three probands have mutations in exons 63–66, three in exons 143 or 144, and ten in exons 167–177. Three of these probands have both of their mutations in alternatively spliced exons. Further studies on the expression of alternatively spliced exons in different muscles combined with the clinical data of the patients with mutations in alternatively spliced exons might provide insights into the role and importance of these exons in different muscles and developmental stages, and explain some phenotypic features. The work is ongoing.