Browsing by Subject "Nemaline myopathy"

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  • Lehtokari, Vilma-Lotta; Gardberg, Maria; Pelin, Katarina; Wallgren-Pettersson, Carina (2018)
    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.
  • Sofieva, Svetlana (Helsingin yliopisto, 2019)
    Nemaline myopathy (NM) is a rare congenital disorder, the most common of congenital myopathies. It affects primarily the skeletal muscles and it is recognised by nemaline bodies in muscle tissue samples and muscle weakness. Mutation of eleven genes are known to lead to NM and the most frequent disease-causing variants are either recessive NEB variants or dominant ACTA1 variants. Variants in NEB are thought to be well tolerated and only 7% of them are hypothesized to be pathogenic. Over 200 pathogenic NEB-variants have been identified in Helsinki and the majority occurred in patients as a combination of two different variants. The missense variants were speculated to have a modifying effect on pathogenicity by affecting nebulin-actin or nebulin-tropomyosin interactions. Nebulin is a gigantic protein coded by NEB and is one of the largest proteins in vertebrates. It is located in the thin filament of the skeletal muscle sarcomere. Enclosed by terminal regions, nebulin has an extensive repetitive modular region that covers over 90% of the protein. The repetitive zone comprises of 26 modules called super repeats (SR). SRs consist of seven simple repeats. There are seven conserved SDXXYK actin-binding sites at each super repeat, one per simple repeat, and one conserved WLKGIGW tropomyosin-binding site. Due to its enormous size and highly repetitive sequence, nebulin is one of the least studied proteins in vivo, in vitro or in silico. In the NM patient database used for this study, there are 70 families with verified pathogenic mutations and in 30 of them, there were additional missense variants in NEB. These missense variants can be pathogenic modifying factors or have no impact on the phenotype. Seven missense variants were selected to study the effect of these mutations on actin-binding capacity compared to wild-type nebulin using the SR panel constructed previously by Laitila and Lehtonen. Also, due to the differences in actin-binding capacity of SRs compared to each other, one of the aims was to determine whether corresponding mutations in different SRs would have a similar or different effect on actin-binding capacity. For this aim, one missense mutation in the strongly actin-binding SR 1, and one in the weakly actin-binding SR 7 were selected from the NM database, and corresponding variants were created. Also, an in-frame deletion in SR7 found in the ExAC database and the corresponding mutation in SR1 were constructed for this study. The actin-binding strength was determined using actin co-sedimentation assay and actin affinity assay. The results for co-sedimentation assay indicate that missense variants can have an effect on nebulin-actin interactions and, therefore, can be a possible cause for NM. The corresponding mutations had no correlation in their effect on actin-binding strength, just the opposite. S1-m-2 decreased actin-binding strength of SR1 and S7-m-2 had no effect on SR7. Likewise, S7-m-1 and S7-del-1 decreased actin-binding strength of SR7 and corresponding mutations had no effect on SR1. The selected missense mutations found in NM patients in SRs 2 and 4 decreased actin-binding strength, if located at the actin-binding sites and in SR 10 increased the actin-binding strength, if located at the actin-binding site. The change in actin binding strength was defined as significant if the P-value was below 0.005. The more accurate affinity assay was performed as a trial only for S16 and S16-m-1, a variant at a tropomyosin-binding site close to an actin-binding site. It indicated a difference in actin-binding affinity missed by the actin co-sedimentation assay. The results are preliminary, but show big promise and should be optimized and implemented in the future missense mutation affinity studies. In an attempt to understand if the effect missense mutations have on nebulin-actin interaction is based on the change in nebulin structure, the 3D-structure of each produced fusion protein was predicted in silico. Considering that the variants were produced as GST-fusion proteins, the position and effect of GST in them is also a point of interest. In order to predict the structure of these large proteins, a combined approach was implemented using I-TASSER (Iterative Threading ASSEmbly Refinement) software. The software uses ab initio modeling, threading methods and atomic-level structure refinement to build an accurate 3D-model of a protein from sequence. According to the predicted 3D models of the fusion proteins, the GST-part of the proteins folds into a globular structure and acts as a core around which the nebulin fragments fold. The GST does not bind to actin and is positioned on the inside, which indicates minimal effect on nebulin-actin interaction, but may be a reason for an alternative nebulin fragment folding. The accuracy of the default set of programs in software does not give the definitive answer of the possible effect missense mutations can have on structural changes. However, I-TASSER approach for 3D-modeling is promising with further software optimization and can possibly serve as an effective bioinformatic tool in the future.
  • Ross, Jacob A.; Levy, Yotam; Ripolone, Michela; Kolb, Justin S.; Turmaine, Mark; Holt, Mark; Lindqvist, Johan; Claeys, Kristl G.; Weis, Joachim; Monforte, Mauro; Tasca, Giorgio; Moggio, Maurizio; Figeac, Nicolas; Zammit, Peter S.; Jungbluth, Heinz; Fiorillo, Chiara; Vissing, John; Witting, Nanna; Granzier, Henk; Zanoteli, Edmar; Hardeman, Edna C.; Wallgren-Pettersson, Carina; Ochala, Julien (2019)
    Nemaline myopathy (NM) is a skeletal muscle disorder caused by mutations in genes that are generally involved in muscle contraction, in particular those related to the structure and/or regulation of the thin filament. Many pathogenic aspects of this disease remain largely unclear. Here, we report novel pathological defects in skeletal muscle fibres of mouse models and patients with NM: irregular spacing and morphology of nuclei; disrupted nuclear envelope; altered chromatin arrangement; and disorganisation of the cortical cytoskeleton. Impairments in contractility are the primary cause of these nuclear defects. We also establish the role of microtubule organisation in determining nuclear morphology, a phenomenon which is likely to contribute to nuclear alterations in this disease. Our results overlap with findings in diseases caused directly by mutations in nuclear envelope or cytoskeletal proteins. Given the important role of nuclear shape and envelope in regulating gene expression, and the cytoskeleton in maintaining muscle fibre integrity, our findings are likely to explain some of the hallmarks of NM, including contractile filament disarray, altered mechanical properties and broad transcriptional alterations.
  • Laitila, Jenni M.; McNamara, Elyshia L.; Wingate, Catherine D.; Goullee, Hayley; Ross, Jacob A.; Taylor, Rhonda L.; van der Pijl, Robbert; Griffiths, Lisa M.; Harries, Rachel; Ravenscroft, Gianina; Clayton, Joshua S.; Sewry, Caroline; Lawlor, Michael W.; Ottenheijm, Coen A. C.; Bakker, Anthony J.; Ochala, Julien; Laing, Nigel G.; Wallgren-Pettersson, Carina; Pelin, Katarina; Nowak, Kristen J. (2020)
    Nemaline myopathy (NM) caused by mutations in the gene encoding nebulin (NEB) accounts for at least 50% of all NM cases worldwide, representing a significant disease burden. Most NEB-NM patients have autosomal recessive disease due to a compound heterozygous genotype. Of the few murine models developed for NEB-NM, most are Neb knockout models rather than harbouring Neb mutations. Additionally, some models have a very severe phenotype that limits their application for evaluating disease progression and potential therapies. No existing murine models possess compound heterozygous Neb mutations that reflect the genotype and resulting phenotype present in most patients. We aimed to develop a murine model that more closely matched the underlying genetics of NEB-NM, which could assist elucidation of the pathogenetic mechanisms underlying the disease. Here, we have characterised a mouse strain with compound heterozygous Neb mutations; one missense (p.Tyr2303His), affecting a conserved actin-binding site and one nonsense mutation (p.Tyr935*), introducing a premature stop codon early in the protein. Our studies reveal that this compound heterozygous model, Neb(Y2303H, Y935X), has striking skeletal muscle pathology including nemaline bodies. In vitro whole muscle and single myofibre physiology studies also demonstrate functional perturbations. However, no reduction in lifespan was noted. Therefore, Neb(Y2303H,Y935X) mice recapitulate human NEB-NM and are a much needed addition to the NEB-NM mouse model collection. The moderate phenotype also makes this an appropriate model for studying NEB-NM pathogenesis, and could potentially be suitable for testing therapeutic applications.
  • Laitila, Jenni M; McNamara, Elyshia L; Wingate, Catherine D; Goullee, Hayley; Ross, Jacob A; Taylor, Rhonda L; van der Pijl, Robbert; Griffiths, Lisa M; Harries, Rachel; Ravenscroft, Gianina; Clayton, Joshua S; Sewry, Caroline; Lawlor, Michael W; Ottenheijm, Coen A C; Bakker, Anthony J; Ochala, Julien; Laing, Nigel G; Wallgren-Pettersson, Carina; Pelin, Katarina; Nowak, Kristen J (BioMed Central, 2020)
    Abstract Nemaline myopathy (NM) caused by mutations in the gene encoding nebulin (NEB) accounts for at least 50% of all NM cases worldwide, representing a significant disease burden. Most NEB-NM patients have autosomal recessive disease due to a compound heterozygous genotype. Of the few murine models developed for NEB-NM, most are Neb knockout models rather than harbouring Neb mutations. Additionally, some models have a very severe phenotype that limits their application for evaluating disease progression and potential therapies. No existing murine models possess compound heterozygous Neb mutations that reflect the genotype and resulting phenotype present in most patients. We aimed to develop a murine model that more closely matched the underlying genetics of NEB-NM, which could assist elucidation of the pathogenetic mechanisms underlying the disease. Here, we have characterised a mouse strain with compound heterozygous Neb mutations; one missense (p.Tyr2303His), affecting a conserved actin-binding site and one nonsense mutation (p.Tyr935*), introducing a premature stop codon early in the protein. Our studies reveal that this compound heterozygous model, NebY2303H, Y935X, has striking skeletal muscle pathology including nemaline bodies. In vitro whole muscle and single myofibre physiology studies also demonstrate functional perturbations. However, no reduction in lifespan was noted. Therefore, NebY2303H,Y935X mice recapitulate human NEB-NM and are a much needed addition to the NEB-NM mouse model collection. The moderate phenotype also makes this an appropriate model for studying NEB-NM pathogenesis, and could potentially be suitable for testing therapeutic applications.
  • Sewry, Caroline A.; Laitila, Jenni M.; Wallgren-Pettersson, Carina (2019)
    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.