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Pathogenetic Mechanisms and Genotype–Phenotype Correlations in Nemaline Myopathies and Related Disorders Caused by Mutations in Tropomyosin Genes and Nebulin

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Pathogenetic Mechanisms and Genotype–Phenotype Correlations in Nemaline Myopathies and Related Disorders Caused by Mutations in Tropomyosin Genes and Nebulin Pathogenetic mechanisms and genotype–phenotype correlations in nemaline myopathies and related disorders caused by mutations in tropomyosin genes and nebulin Minttu Marttila University of Helsinki 2014 Pathogenetic mechanisms and genotype–phenotype correlations in nemaline myopathies and related disorders caused by mutations in tropomyosin genes and nebulin Minttu Marttila The department of Medical Genetics, University of Helsinki, Helsinki, Finland and The Folkhälsan Institute of Genetics Academic Dissertation To be presented, with the permission of the Faculty of Biological and Environmental Sciences of University of Helsinki, for public examination in the Lecture Hall 1, Biomedicum Helsinki, Haartmaninkatu 8, Helsinki on 7th of November at 12 o’clock Helsinki 2014 Supervisors Associate professor Carina Wallgren-Pettersson, DM The Folkhälsan Institute of Genetics and Department of Medical Genetics University of Helsinki Finland Associate professor Mikaela Grönholm, PhD Division of Biochemistry Department of Biological and Environmental Sciences University of Helsinki Finland Reviewers Associate professor Pirta Hotulainen, PhD Neuroscience Center University of Helsinki Finland Associate professor Jukka Moilanen, DM Department of Clinical Genetics Oulu University Hospital Finland Opponent Julien Ochala, PhD Centre of Human and Aerospace Physiological Sciences King's College London UK ISBN 978-951-51-0287-4 (paperback) ISBN 978-951-51-0288-1 (PDF) University of Helsinki Print Helsinki 2014 LIST OF ORIGINAL PUBLICATIONS The thesis is based on the following publications. I Marttila M, Lemola E, Wallefeld W, Memo M, Donner K, Laing NG, Marston S, Grönholm M, Wallgren-Pettersson C. Abnormal actin binding of aberrant β-tropomyosins is a molecular cause of muscle weakness in TPM2-related nemaline and cap myopathy. Biochem J 2012 15;442(1):231-9. II Marttila M, Lehtokari VL, Marston S, Nyman TA, Barnerias C, Beggs AH, Bertini E, Ceyhan-Birsoy O, Cintas P, Gerard M, Gilbert-Dussardier B, Hogue JS, Longman C, Eymard B, Frydman M, Kang PB, Klinge L, Kolski H, Lochmüller H, Magy L, Manel V, Mayer M, Mercuri E, North KN, Peudenier-Robert S, Pihko H, Probst FJ, Reisin R, Stewart W, Taratuto AL, de Visser M, Wilichowski E, Winer J, Nowak K, Laing NG, Winder TL, Monnier N, Clarke NF, Pelin K, Grönholm M, Wallgren-Pettersson C. Mutation update and genotype- phenotype correlations of novel and previously described mutations in TPM2 and TPM3 causing congenital myopathies. Hum Mutat 2014 35(7):779-90. III Marttila M*, Hanif M*, Lemola E, Nowak KJ, Laitila J, Grönholm M, Wallgren-Pettersson C, Pelin K. Nebulin interactions with actin and tropomyosin are altered by disease-causing mutations. Skelet Muscle 2014 1(4)15. *The authors contributed equally to the work. The articles are reprinted with the permission of the copyright owners. Contents List of original publications Author contributions Abbreviations Abstract Tiivistelmä Introduction Review of the literature 1 1 SKELETAL MUSCLE 1 1.1 Muscle fibre types 1 1.2 The Muscle sarcomere 2 2 Congenital myopathies 15 2.1 Nemaline myopathies 16 2.2 Disorders related to nemaline myopathy 24 2 Aims of the study 27 3 Materials and methods 27 3.1 Polymerase Chain Reaction (PCR) and sequencing 27 3.2 Constructs 27 3.3 Production of wild-type and aberrant β-tropomyosins 28 3.4 RNA isolation and reverse transcriptase polymerase chain reaction (RT-PCR) 28 3.5 In vitro mutagenesis and sequencing 29 3.6 Construction of vectors for the expression of nebulin super-repeats 31 3.7 Nebulin production in Escherichia coli 31 3.8 Three-dimensional models 32 4 RESULTS AND DISCUSSION 33 4.1 Novel mutations in TPM2 and TPM3 33 4.2 Clinical correlations 41 4.3 Genotype-phenotype correlations 43 4.4 Identification of novel phosphorylation sites in β-tropomyosin 49 4.5 Functional analysis by in vitro motility assay 50 4.6 Mass spectrometry and three-dimensional models 51 4.7 Circular dichroism spectra of β-tropomyosin 53 4.8 Nebulin interactions with actin and tropomyosin 55 4.9 Conclusions and future prospects 59 5 Acknowledgements 61 6 References 63 AUTHOR CONTRIBUTIONS I CWP was responsible for planning the project, for clinical correlations and contributed to drafting the text. MM performed actin-binding experiments, three-dimensional models, mass- spectrometric analysis and contributed to writing the article. EL was responsible for protein production and actin-binding experiments. KD produced three tropomyosin constructs. MG, a senior protein expert, planned parts of the experimental work and wrote parts of the paper. NL and SM contributed to writing the paper. WW performed circular dichroism experiments and drafted parts of the text. MMemo performed in vitro motility experiments and contributed to writing the article. II MM was responsible for coordinating the collection of mutations and writing the paper. VLL contributed to writing the article and performed the mutation detection using dHPLC and sequencing. TAN performed the mass-spectrometric analyses. KP performed the pathogenicity analysis and wrote parts of the paper. MG and SM contributed to writing the article. CWP reported the clinical correlations and coordinated the project. The remaining authors contributed to the article by donating mutations and clinical data discovered in their laboratories. III MH performed cloning and site-directed mutagenesis. Nebulin protein fragments were produced by MH and MM. Tropomyosins were produced and purified by EL and KJN. MH performed the nebulin-actin binding experiments together with EL. MM studied wild type (wt) and mutant nebulin binding to wt tropomyosins. KP and JL produced the figure on nebulin super repeats. MM, MH, KP, MG and CWP planned the study and wrote the article. ABBREVIATIONS ACTA1 slow skeletal muscle -actin ATP adenosine triphosphate bp base pair BTB–BACK bric-a-brac, tram-track, broad-complex –BTB and C-terminal kelch Ca2+ calsium ion CD circular dicroism cDNA complimentary DNA CFL2 gene encoding cofilin 2 CFTD congenital fibre-type disproportion DA distal arthrogryposis DHPR dihydropyridine receptor L-type Ca2+ channel DNA deoxyribonucleic acid EC excitation–contraction coupling e.g. exempli grafia EMG electromyography F-actin filamentous actin FSD fibre size disproortion GST glutathione-S-transferase H&E Hematoxylin&eosin i.e. id est IPTG isopropyl-β-D-thiogalactopyranoside kb kilobase KBTBD13 a member of the BTB/kelch protein family kDa kiloDalton KLHL40 and kelch-like family members 40 and 41 KLHL41 KO knock-out LB Luria-Bertani media LMOD3 gene encoding leiomodin-3 Mg2+ magnesium ion mM milli molaarinen μM mikro molaarinen µg mikro gramma µl mikro litra NEB gene encoding nebulin NM nemaline myopathy MDa MegaDalton MYH gene encoding myosin heavy-chain pCa negative logarithm of the concentration of calcium ions in solution PDB protein data bank PBS phosphate buffered saline RT-PCR reverse transcriptase polymerase chain reaction RYR1 gene encoding ryanodine receptor 1 RyR1 the ryanodine receptor Ca2+ release channel SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis Sf9 Spodoptera frugiperda hyönteissolut SR sarcoplasmic reticulum Tm tropomyosin TnC troponin C TnI troponin I TnT troponin T TNNT1 gene encoding troponiini T TPM2 gene encoding β-tropomyosin TPM3 gene encoding slow α-tropomyosin UTR untranslated region VL vastus lateralis wt wild type ABSTRACT This thesis project aimed to collect all mutations in TPM2 and TPM3 genes hitherto found to cause congenital myopathies, to perform genotype-phenotype correlations, and to increase our understanding of the pathogenetic mechanisms of congenital myopathies caused by mutations in the tropomyosin and nebulin genes. Nemaline myopathy (NM), a rare, genetic muscle disorder defined on the basis of muscle dysfunction and the presence of structural abnormalities in the muscle fibres (i.e. nemaline bodies), is caused by mutations in ten genes known to date: Nebulin (NEB), α-actin (ACTA1), α-tropomyosin (TPM3), β-tropomyosin (TPM2), troponin T (TNNT1), cofilin 2 (CFL2), KBTBD13, KLHL40, KLHL41 and leiomodin 3 (LMOD 3). This study concentrated on the investigation of β-tropomyosin and nebulin since both have been identified by our group as genes causative of NM. In addition, this study focused on α-tropomyosin because it forms dimers with β-tropomyosin. Tropomyosin controls muscle contraction by inhibiting the actin–myosin interaction in a calcium-sensitive manner. Mutations in tropomyosin genes may cause NM, cap myopathy, congenital fibre-type disproportion (CFTD), distal arthrogryposes (DA) and Escobar syndrome. We correlated the clinical picture of these diseases to novel and previously published mutations to the TPM2 and TPM3 genes, including 30 mutations causing amino acid changes in TPM2 and 20 mutations in TPM3. Most mutations were heterozygous changes associated with autosomal dominant disease including 19 novel and 31 previously reported mutations. Previous studies found that five mutations in TPM2 and one in TPM3 caused an increased Ca2+ sensitivity, resulting in a hypercontractile molecular phenotype. Patients with hypercontractile molecular phenotypes more often had contractures of the limb joints (18/19) and jaw (6/19) than those with non-hypercontractile molecular phenotypes (2/22 and 1/22). Our in silico predictions show that most tropomyosin mutations affect the tropomyosin– actin association or tropomyosin head-to-tail binding. We studied the pathogenetic mechanisms to which five disease-causing mutations in β-tropomyosin
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