Novel mutations in NEB cause abnormal nebulin expression and markedly impaired muscle force generation in severe nemaline myopathy. Skelet muscle 1:23

Division of Genetics and Program in Genomics, The Manton Center for Orphan Disease Research, Children's Hospital Boston, Harvard Medical School, 300 Longwood Avenue, CLSB 15026, Boston, MA 02115, USA.
Skeletal Muscle 06/2011; 1(1):23. DOI: 10.1186/2044-5040-1-23
Source: PubMed


Nemaline myopathy (NM) is a congenital muscle disease associated with weakness and the presence of nemaline bodies (rods) in muscle fibers. Mutations in seven genes have been associated with NM, but the most commonly mutated gene is nebulin (NEB), which is thought to account for roughly 50% of cases.
We describe two siblings with severe NM, arthrogryposis and neonatal death caused by two novel NEB mutations: a point mutation in intron 13 and a frameshift mutation in exon 81. Levels of detectable nebulin protein were significantly lower than those in normal control muscle biopsies or those from patients with less severe NM due to deletion of NEB exon 55. Mechanical studies of skinned myofibers revealed marked impairment of force development, with an increase in tension cost.
Our findings demonstrate that the mechanical phenotype of severe NM is the consequence of mutations that severely reduce nebulin protein levels and suggest that the level of nebulin expression may correlate with the severity of disease.

Download full-text


Available from: Henk L Granzier,
  • Source
    • "Nebulin-deficient myofibrils also have a reduced Ca 2+ sensitivity of force production (Witt et al., 2006), a slower rate of tension redevelopment and an elevated tension cost, meaning more ATP has to be expended to generate a given tension. This suggests that there is a reduced fraction of force-generating cross-bridges, which, in turn, contributes to muscle weakness (Ottenheijm et al., 2010; Lawlor et al., 2011a). Recently studies have shown that in vitro treatment of nebulin-deficient myofibres with a fast skeletal muscle troponin activator (CK-2066260) enhanced force production at submaximal activating Ca 2+ levels (de Winter et al., 2013; Lee et al., 2013; Ottenheijm et al., 2013). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The congenital myopathies are a diverse group of genetic skeletal muscle diseases, which typically present at birth or in early infancy. There are multiple modes of inheritance and degrees of severity (ranging from foetal akinesia, through lethality in the newborn period to milder early and later onset cases). Classically, the congenital myopathies are defined by skeletal muscle dysfunction and a non-dystrophic muscle biopsy with the presence of one or more characteristic histological features. However, mutations in multiple different genes can cause the same pathology and mutations in the same gene can cause multiple different pathologies. This is becoming ever more apparent now that, with the increasing use of next generation sequencing, a genetic diagnosis is achieved for a greater number of patients. Thus, considerable genetic and pathological overlap is emerging, blurring the classically established boundaries. At the same time, some of the pathophysiological concepts underlying the congenital myopathies are moving into sharper focus. Here we explore whether our emerging understanding of disease pathogenesis and underlying pathophysiological mechanisms, rather than a strictly gene-centric approach, will provide grounds for a different and perhaps complementary grouping of the congenital myopathies, that at the same time could help instil the development of shared potential therapeutic approaches. Stemming from recent advances in the congenital myopathy field, five key pathophysiology themes have emerged: defects in (i) sarcolemmal and intracellular membrane remodelling and excitation-contraction coupling; (ii) mitochondrial distribution and function; (iii) myofibrillar force generation; (iv) atrophy; and (v) autophagy. Based on numerous emerging lines of evidence from recent studies in cell lines and patient tissues, mouse models and zebrafish highlighting these unifying pathophysiological themes, here we review the congenital myopathies in relation to these emerging pathophysiological concepts, highlighting both areas of overlap between established entities, as well as areas of distinction within single gene disorders. Published by Oxford University Press on behalf of the Guarantors of Brain 2014. This work is written by US Government employees and is in the public domain in the US.
    Brain 12/2014; 138(2). DOI:10.1093/brain/awu368 · 9.20 Impact Factor
  • Source
    • "Recent studies have shown that patients with NM caused by mutations in NEB may have markedly lower levels of nebulin protein in their muscles than healthy individuals, leading to lower calcium sensitivity of force generation [37,39,41]. A lower abundance of nebulin has been associated with the in-frame deletion of exon 55 (p.Arg2478_Asp2512del) included in the present study, as well as with frameshift and splice-site mutations in NEB[39,41]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background Nemaline myopathy (NM) is a rare genetic muscle disorder, but one of the most common among the congenital myopathies. NM is caused by mutations in at least nine genes: Nebulin (NEB), α-actin (ACTA1), α-tropomyosin (TPM3), β-tropomyosin (TPM2), troponin T (TNNT1), cofilin-2 (CFL2), Kelch repeat and BTB (POZ) domain-containing 13 (KBTBD13), and Kelch-like family members 40 and 41 (KLHL40 and KLHL41). Nebulin is a giant (600 to 900 kDa) filamentous protein constituting part of the skeletal muscle thin filament. Around 90% of the primary structure of nebulin is composed of approximately 35-residue α-helical domains, which form super repeats that bind actin with high affinity. Each super repeat has been proposed to harbor one tropomyosin-binding site. Methods We produced four wild-type (WT) nebulin super repeats (S9, S14, S18, and S22), 283 to 347 amino acids long, and five corresponding repeats with a patient mutation included: three missense mutations (p.Glu2431Lys, p.Ser6366Ile, and p.Thr7382Pro) and two in-frame deletions (p.Arg2478_Asp2512del and p.Val3924_Asn3929del). We performed F-actin and tropomyosin-binding experiments for the nebulin super repeats, using co-sedimentation and GST (glutathione-S-transferase) pull-down assays. We also used the GST pull-down assay to test the affinity of WT nebulin super repeats for WT α- and β–tropomyosin, and for β-tropomyosin with six patient mutations: p.Lys7del, p.Glu41Lys, p.Lys49del, p.Glu117Lys, p.Glu139del and p.Gln147Pro. Results WT nebulin was shown to interact with actin and tropomyosin. Both the nebulin super repeats containing the p.Glu2431Lys mutation and nebulin super repeats lacking exon 55 (p.Arg2478_Asp2512del) showed weak affinity for F-actin compared with WT fragments. Super repeats containing the p.Ser6366Ile mutation showed strong affinity for actin. When tested for tropomyosin affinity, super repeats containing the p.Glu2431Lys mutation showed stronger binding than WT proteins to tropomyosin, and the super repeat containing the p.Thr7382Pro mutation showed weaker binding than WT proteins to tropomyosin. Super repeats containing the deletion p.Val3924_Asn3929del showed similar affinity for actin and tropomyosin as that seen with WT super repeats. Of the tropomyosin mutations, only p.Glu41Lys showed weaker affinity for nebulin (super repeat 18). Conclusions We demonstrate for the first time the existence of direct tropomyosin-nebulin interactions in vitro, and show that nebulin interactions with actin and tropomyosin are altered by disease-causing mutations in nebulin and tropomyosin.
    Skeletal Muscle 08/2014; 4(1):15. DOI:10.1186/2044-5040-4-15
  • Source
    • "Patients from families 1 and 2 died from a severe myopathy shortly after birth, and both were found to harbour NEB mutations. NEB codes for the contractile unit scaffold protein nebulin and mutations in NEB usually give rise to a mild childhood or adult onset myopathy, but also rare fatal cases as for our patients have been reported [19], [20], [21]. The patients from families 3, 4, 5 and 6 were all found to carry mutations in RYR1, although they were clinically different. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Congenital myopathies are severe muscle disorders affecting adults as well as children in all populations. The diagnosis of congenital myopathies is constrained by strong clinical and genetic heterogeneity. Moreover, the majority of patients present with unspecific histological features, precluding purposive molecular diagnosis and demonstrating the need for an alternative and more efficient diagnostic approach. We used exome sequencing complemented by histological and ultrastructural analysis of muscle biopsies to identify the causative mutations in eight patients with clinically different skeletal muscle pathologies, ranging from a fatal neonatal myopathy to a mild and slowly progressive myopathy with adult onset. We identified RYR1 (ryanodine receptor) mutations in six patients and NEB (nebulin) mutations in two patients. We found novel missense and nonsense mutations, unraveled small insertions/deletions and confirmed their impact on splicing and mRNA/protein stability. Histological and ultrastructural findings of the muscle biopsies of the patients validated the exome sequencing results. We provide the evidence that an integrated strategy combining exome sequencing with clinical and histopathological investigations overcomes the limitations of the individual approaches to allow a fast and efficient diagnosis, accelerating the patient's access to a better healthcare and disease management. This is of particular interest for the diagnosis of congenital myopathies, which involve very large genes like RYR1 and NEB as well as genetic and phenotypic heterogeneity.
    PLoS ONE 06/2013; 8(6):e67527. DOI:10.1371/journal.pone.0067527 · 3.23 Impact Factor
Show more