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Nemaline myopathies are a heterogenous group of congenital myopathies caused by de novo, dominantly or recessively inherited mutations in at least twelve genes. The genes encoding skeletal α-actin (ACTA1) and nebulin (NEB) are the commonest genetic cause. Most patients have congenital onset characterized by muscle weakness and hypotonia, but the spectrum of clinical phenotypes is broad, ranging from severe neonatal presentations to onset of a milder disorder in childhood. Most patients with adult onset have an autoimmune-related myopathy with a progressive course. The wide application of massively parallel sequencing methods is increasing the number of known causative genes and broadening the range of clinical phenotypes. Nemaline myopathies are identified by the presence of structures that are rod-like or ovoid in shape with electron microscopy, and with light microscopy stain red with the modified Gömöri trichrome technique. These rods or nemaline bodies are derived from Z lines (also known as Z discs or Z disks) and have a similar lattice structure and protein content. Their shape in patients with mutations in KLHL40 and LMOD3 is distinctive and can be useful for diagnosis. The number and distribution of nemaline bodies varies between fibres and different muscles but does not correlate with severity or prognosis. Additional pathological features such as caps, cores and fibre type disproportion are associated with the same genes as those known to cause the presence of rods. Animal models are advancing the understanding of the effects of various mutations in different genes and paving the way for the development of therapies, which at present only manage symptoms and are aimed at maintaining muscle strength, joint mobility, ambulation, respiration and independence in the activities of daily living.

The congenital myopathies form a large clinically and genetically heterogeneous group of disorders. Currently mutations in at least 27 different genes have been reported to cause a congenital myopathy, but the number is expected to increase due to the accelerated use of next-generation sequencing methods. There is substantial overlap between the causative genes and the clinical and histopathologic features of the congenital myopathies. The mode of inheritance can be autosomal recessive, autosomal dominant or X-linked. Both dominant and recessive mutations in the same gene can cause a similar disease phenotype, and the same clinical phenotype can also be caused by mutations in different genes. Clear genotype-phenotype correlations are few and far between., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We present here a Finnish nemaline myopathy family with a dominant mutation in the skeletal muscle α-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., (Copyright © 2017 Elsevier B.V. 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., (© 2014 WILEY PERIODICALS, INC.)

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., (© 2014 WILEY PERIODICALS, INC.)

Nemaline myopathy (NM) is a rare congenital muscle disorder primarily affecting skeletal muscles that results in neonatal death in severe cases as a result of associated respiratory insufficiency. NM is thought to be a disease of sarcomeric thin filaments as six of eight known genes whose mutation can cause NM encode components of that structure, however, recent discoveries of mutations in non-thin filament genes has called this model in question. We performed whole-exome sequencing and have identified recessive small deletions and missense changes in the Kelch-like family member 41 gene (KLHL41) in four individuals from unrelated NM families. Sanger sequencing of 116 unrelated individuals with NM identified compound heterozygous changes in KLHL41 in a fifth family. Mutations in KLHL41 showed a clear phenotype-genotype correlation: Frameshift mutations resulted in severe phenotypes with neonatal death, whereas missense changes resulted in impaired motor function with survival into late childhood and/or early adulthood. Functional studies in zebrafish showed that loss of Klhl41 results in highly diminished motor function and myofibrillar disorganization, with nemaline body formation, the pathological hallmark of NM. These studies expand the genetic heterogeneity of NM and implicate a critical role of BTB-Kelch family members in maintenance of sarcomeric integrity in NM., (Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Centronuclear myopathy (CNM) is a genetically heterogeneous disorder associated with general skeletal muscle weakness, type I fiber predominance and atrophy, and abnormally centralized nuclei. Autosomal dominant CNM is due to mutations in the large GTPase dynamin 2 (DNM2), a mechanochemical enzyme regulating cytoskeleton and membrane trafficking in cells. To date, 40 families with CNM-related DNM2 mutations have been described, and here we report 60 additional families encompassing a broad genotypic and phenotypic spectrum. In total, 18 different mutations are reported in 100 families and our cohort harbors nine known and four new mutations, including the first splice-site mutation. Genotype-phenotype correlation hypotheses are drawn from the published and new data, and allow an efficient screening strategy for molecular diagnosis. In addition to CNM, dissimilar DNM2 mutations are associated with Charcot-Marie-Tooth (CMT) peripheral neuropathy (CMTD1B and CMT2M), suggesting a tissue-specific impact of the mutations. In this study, we discuss the possible clinical overlap of CNM and CMT, and the biological significance of the respective mutations based on the known functions of dynamin 2 and its protein structure. Defects in membrane trafficking due to DNM2 mutations potentially represent a common pathological mechanism in CNM and CMT., (© 2012 Wiley Periodicals, Inc.)

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Myotubular myopathy and centronuclear myopathies (CNM) are congenital myopathies characterized by generalized muscle weakness and mislocalization of muscle fiber nuclei. Genetically distinct forms exist, and mutations in BIN1 were recently identified in autosomal recessive cases (ARCNM). Amphiphysins have been implicated in membrane remodeling in brain and skeletal muscle. Our objective was to decipher the pathogenetic mechanisms underlying different forms of CNM, with a focus on ARCNM cases. In this study, we compare the histopathological features from patients with X-linked, autosomal recessive, and dominant forms, respectively, mutated in myotubularin (MTM1), amphiphysin 2 (BIN1), and dynamin 2 (DNM2). We further characterize the ultrastructural defects in ARCNM muscles. We demonstrate that the two BIN1 isoforms expressed in skeletal muscle possess the phosphoinositide-binding domain and are specifically targeted to the triads close to the DHPR-RYR1 complex. Cardiac isoforms do not contain this domain, suggesting that splicing of BIN1 regulates its specific function in skeletal muscle. Immunofluorescence analyses of muscles from patients with BIN1 mutations reveal aberrations of BIN1 localization and triad organization. These defects are also observed in X-linked and autosomal dominant forms of CNM and in Mtm1 knockout mice. In addition to previously reported implications of BIN1 in cancer as a tumor suppressor, these findings sustain an important role for BIN1 skeletal muscle isoforms in membrane remodeling and organization of the excitation-contraction machinery. We propose that aberrant BIN1 localization and defects in triad structure are part of a common pathogenetic mechanism shared between the three forms of centronuclear myopathies.

The ACTA1 gene encodes skeletal muscle alpha-actin, which is the predominant actin isoform in the sarcomeric thin filaments of adult skeletal muscle, and essential, along with myosin, for muscle contraction. ACTA1 disease-causing mutations were first described in 1999, when a total of 15 mutations were known. In this article we describe 177 different disease-causing ACTA1 mutations, including 85 that have not been described before. ACTA1 mutations result in five overlapping congenital myopathies: nemaline myopathy; intranuclear rod myopathy; actin filament aggregate myopathy; congenital fiber type disproportion; and myopathy with core-like areas. Mixtures of these histopathological phenotypes may be seen in a single biopsy from one patient. Irrespective of the histopathology, the disease is frequently clinically severe, with many patients dying within the first year of life. Most mutations are dominant and most patients have de novo mutations not present in the peripheral blood DNA of either parent. Only 10% of mutations are recessive and they are genetic or functional null mutations. To aid molecular diagnosis and establishing genotype-phenotype correlations, we have developed a locus-specific database for ACTA1 variations (http://waimr.uwa.edu.au).

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.

Beals-Hecht syndrome or congenital contractural arachnodactyly (CCA) is a rare, autosomal dominant connective tissue disorder characterized by crumpled ears, arachnodactyly, contractures, and scoliosis. Recent reports also mention aortic root dilatation, a finding previously thought to differentiate the condition from Marfan syndrome (MFS). In many cases, the condition is caused by mutations in the fibrillin 2 gene (FBN2) with 26 mutations reported so far, all located in the middle region of the gene (exons 23-34). We directly sequenced the entire FBN2 gene in 32 probands clinically diagnosed with CCA. In 14 probands, we found 13 new and one previously described FBN2 mutation including a mutation in exon 17, expanding the region in which FBN2 mutations occur in CCA. Review of the literature showed that the phenotype of the FBN2 positive patients was comparable to all previously published FBN2-positive patients. In our FBN2-positive patients, cardiovascular involvement included mitral valve prolapse in two adult patients and aortic root enlargement in three patients. Whereas the dilatation regressed in one proband, it remained marked in a child proband (z-score: 4.09) and his father (z-score: 2.94), warranting echocardiographic follow-up. We confirm paradoxical patellar laxity and report keratoconus, shoulder muscle hypoplasia, and pyeloureteral junction stenosis as new features. In addition, we illustrate large intrafamilial variability. Finally, the FBN2-negative patients in this cohort were clinically indistinguishable from all published FBN2-positive patients harboring a FBN2 mutation, suggesting locus heterogeneity., (2008 Wiley-Liss, Inc.)

Centronuclear myopathies are characterized by muscle weakness and abnormal centralization of nuclei in muscle fibers not secondary to regeneration. The severe neonatal X-linked form (myotubular myopathy) is due to mutations in the phosphoinositide phosphatase myotubularin (MTM1), whereas mutations in dynamin 2 (DNM2) have been found in some autosomal dominant cases. By direct sequencing of functional candidate genes, we identified homozygous mutations in amphiphysin 2 (BIN1) in three families with autosomal recessive inheritance. Two missense mutations affecting the BAR (Bin1/amphiphysin/RVS167) domain disrupt its membrane tubulation properties in transfected cells, and a partial truncation of the C-terminal SH3 domain abrogates the interaction with DNM2 and its recruitment to the membrane tubules. Our results suggest that mutations in BIN1 cause centronuclear myopathy by interfering with remodeling of T tubules and/or endocytic membranes, and that the functional interaction between BIN1 and DNM2 is necessary for normal muscle function and positioning of nuclei.

We present comparisons of the clinical pictures in a series of 60 patients with nemaline myopathy in whom mutations had been identified in the genes for nebulin or skeletal muscle alpha-actin. In the patients with nebulin mutations, the typical form of nemaline myopathy predominated, while severe, mild or intermediate forms were less frequent. Autosomal recessive inheritance had been verified or appeared likely in all nebulin cases. In the patients with actin mutations, the severe form of nemaline myopathy was the most common, but some had the mild or typical form, and a few showed other associated features such as intranuclear rods or actin accumulation. Most cases were sporadic, but in addition there were instances of both autosomal dominant and autosomal recessive inheritance, while two families showed mosaicism for dominant mutations. Although no specific phenotype was found to be associated with mutations in either gene, clinical and histological features together with pedigree data may be used in guiding mutation detection. Finding the causative mutation(s) determines the mode of inheritance and permits prenatal diagnosis if requested, but will not as such permit prognostication.

Mutations in the skeletal muscle alpha-actin gene (ACTA1) associated with congenital myopathy with excess of thin myofilaments, nemaline myopathy and intranuclear rod myopathy were first described in 1999. At that time, only 15 different missense mutations were known in ACTA1. More than 60 mutations have now been identified. This review analyses this larger spectrum of mutations in ACTA1. It investigates the molecular consequences of the mutations found to date, provides a framework for genotype-phenotype correlation and suggests future studies in light of results of investigation of normal and mutant actin in other systems, notably the actin specific to the indirect flight muscles of Drosophila. The larger series confirms that the majority of ACTA1 mutations are dominant, a small number are recessive and most isolated cases with no previous family history have de novo dominant mutations. The severity of the disease caused ranges from lack of spontaneous movements at birth requiring immediate mechanical ventilation, to mild disease compatible with life to adulthood. Overall, the mutations within ACTA1 are randomly distributed throughout the protein. However, the larger series of mutations now available indicates that there may be clustering of mutations associated with some phenotypes, e.g. actin myopathy. This would suggest that interference with certain actin functions may be more associated with certain phenotypes, though the exact pathophysiology of the actin mutations remains unknown.

Previously, we reported results indicating that nebulin was the gene causing the typical form of autosomal recessive nemaline (rod) myopathy. Here we describe the identification of mutations in the nebulin gene in seven offspring of five families affected by the severe congenital form of nemaline myopathy. One pregnancy was terminated on the grounds of foetal abnormality, while six affected infants died at ages ranging from the first day of life to 19 months. Only three of the six neonates were able to establish spontaneous respiration. Three had arthrogryposis. In three of the five families, the mutations were located in exon 184. These mutations are predicted to cause absence of the C-terminal part of nebulin.

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

Nemaline myopathy is a clinically and genetically heterogeneous muscle disorder. In the nebulin gene we have detected a number of autosomal recessive mutations. Both autosomal dominant and recessive mutations have been detected in the genes for alpha-actin and alpha-tropomyosin 3. A recessive mutation causing nemaline myopathy among the Old Order Amish has recently been identified in the gene for slow skeletal muscle troponin T. As linkage studies had shown that at least one further gene exists for nemaline myopathy, we investigated another tropomyosin gene expressed in skeletal muscle, the beta-tropomyosin 2 gene. Screening 66 unrelated patients, using single strand conformation polymorphism analysis and sequencing, we found four polymorphisms and two heterozygous missense mutations. Both mutations affect conserved amino acids, and in both cases, the mutant allele is expressed. We speculate that the observed mutations affect the formation of the tropomyosin dimer and its actin-binding properties.

Nemaline myopathy is a structural congenital myopathy which may show both autosomal dominant and autosomal recessive inheritance patterns. Mutations in three different genes have been identified as the cause of nemaline myopathy: the gene for slow alpha-tropomyosin 3 (TPM3) at 1q22-23, the nebulin gene (NEB) at 2q21.1-q22, and the actin gene (ACTA1) at 1q42. The typical autosomal recessive form appears to be the most common one and is caused by mutations in the nebulin gene. We have studied the pattern of nebulin labeling, in patients with the typical congenital form (ten patients), the severe congenital form (two patients) or the mild, childhood-onset form (one patient), using antibodies against three different domains of nebulin. A qualitative and quantitative nebulin analysis in muscle tissue showed the presence of nebulin in myofibers from all patients. Some differences relating to the rod structure were observed. The majority of the largest subsarcolemmal rods were not labeled with the N2 nebulin antibody (I-band epitope) and showed an indistinct pattern with the two antibodies directed to the Z-band portion of nebulin (epitopes M176-181 and serine-rich domain). Diffuse rods were not revealed using the three antibodies. A discordant pattern of nebulin N2 epitope labeling was found in two affected sisters with a mutation in the nebulin gene, suggesting that modifications in nebulin distribution inside the rods might occur with the progression of the disease. Western blot analysis showed no direct correlation with immunofluorescence data. In nine patients, the band had a molecular weight comparable to the normal control, while in one patient, it was detected with a higher molecular weight. Our results suggest that presence/absence of specific nebulin Z-band epitopes in rod structures is variable and could depend on the degree of rod organization.

X-linked myotubular myopathy (XLMTM; MIM# 310400) is a severe congenital muscle disorder caused by mutations in the MTM1 gene. This gene encodes a dual-specificity phosphatase named myotubularin, defining a large gene family highly conserved through evolution (which includes the putative anti-phosphatase Sbf1/hMTMR5). We report 29 mutations in novel cases, including 16 mutations not described before. To date, 198 mutations have been identified in unrelated families, accounting for 133 different disease-associated mutations which are widespread throughout the gene. Most point mutations are truncating, but 26% (35/133) are missense mutations affecting residues conserved in the Drosophila ortholog and in the homologous MTMR1 gene. Three recurrent mutations affect 17% of the patients, and a total of 21 different mutations were found in several independent families. The frequency of female carriers appears higher than expected (only 17% are de novo mutations). While most truncating mutations cause the severe and early lethal phenotype, some missense mutations are associated with milder forms and prolonged survival (up to 54 years)., (Copyright 2000 Wiley-Liss, Inc.)

The nemaline myopathies are muscle disorders of variable severity and age of onset, with characteristic nemaline bodies in the sarcoplasm. Genes for dominant (NEM1) and recessive (NEM2A) nemaline myopathy have been localised to chromosomes one and two, respectively. A missense mutation in the alpha-tropomyosin gene (TPM3) has been associated with NEM1 in one family. Probands from 76 other nemaline myopathy families have now been screened for TPM3 mutations. One proband, who was not noted to have any weakness neonatally, but who died at 21 months of age, was shown to be homozygous for a single strand conformation polymorphism (SSCP) in skeletal-muscle-specific exon 1 of TPM3. Sequencing revealed homozygosity for a nonsense mutation at codon 31 (CAG to TAG). The patient should have no functioning alpha-tropomyosin slow protein. The nemaline bodies in this patient were exclusively in type one fibres, consistent with the expression of TPM3 only in type one fibres.

Using five monoclonal antibodies against different parts of the dystrophin molecule, we have studied the dystrophin composition of 17 dystrophin-positive fibres in a muscle biopsy from a boy with Xp21 muscular dystrophy of Duchenne-type severity. The fibres showed five distinct, reproducible, immunoreactive dystrophin profiles. All the profiles included both the N-terminal and the C-terminal domains, but between these domains, different fibres were negative for different antibodies, suggesting the somatic loss of certain exons. We interpret this as the first in situ evidence of an individual having different patterns of missing exons leading to restoration of the reading frame in various ways in the original germline frame-shifting deletion of exons 35-43. It follows that various somatic mutations had taken place in different fibres.