Functional studies of YBX3 variants associated with nemaline myopathy

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Abstracts 2016 / Neuromuscular Disorders 26 (2016) S88–S212

P.155 Contractile weakness in NEM3 patients is caused by dysfunctional sarcomeres B. Joureau 1, J. de Winter 1, S. Conijn 1, E. Malfatti 2, M. Yuen 3, N. Clarke 3, N. Romero 2, D. Rassier 4, A. Beggs 5, C. Ottenheijm 1 1 VU University Medical Center, Amsterdam, Netherlands; 2 INSERM, Paris, France; 3 Institution for Neuroscience and Muscle Research, Sydney, Australia; 4 Physics and Physiology, McGill University, Montréal, Canada; 5 The Manton Center for Orphan Disease Research, Children’s Hospital Boston, Boston, USA

c.4828G > A, p.Glu1610Lys affecting the MYH7 mid-rod domain. The p.Glu1610Lys, predicted to be damaging, affects a conserved amino acid located in the MYH7 rod-tail domain, a region rarely mutated in patients with a combined cardiac and skeletal muscle involvement. By contrast these clinical and histological presentations have never been described before in association with MYH7 defects. Our study significantly broadens the clinical and pathological spectrum of MYH7-related disorders and suggests screening MYH7 gene in adult-onset nemaline myopathies with cardiomyopathy.

Nemaline myopathy is a heterogeneous congenital non-dystrophic myopathy, characterized by muscle weakness and the presence of nemaline rods in muscle fibers. The pathophysiology of muscle weakness in nemaline myopathy caused by mutations in ACTA1 (NEM3) is incompletely understood. ACTA1 encodes skeletal muscle α-actin, the main component of the sarcomeric thin filament. Hence, we hypothesized that muscle weakness in NEM3 is caused by sarcomeric dysfunction. Contractile function of permeabilized muscle fibers (SF) and of single myofibrils (MF) was determined in control (CTRL, n = 6) and NEM3 (n = 14) subjects. SF and MF were isolated and mounted between a force transducer and length motor. At sarcomere length of 2.5 μm, by exposure to [Ca2+] solutions, maximal tension, cross bridge cycling kinetics (Ktr) and the active stiffness were determined. Maximal tension at incremental sarcomere lengths (range 2.0–3.5 μm) revealed the force–sarcomere length relationship and by fitting to a 2nd order polynomial the sarcomere length at maximal force (SLopt) and the sarcomere length at 50% of maximal force (SL50) were obtained. Data: mean ± SEM. In NEM3 patients, the contractility of SF was significantly decreased compared to CTRL: maximal active tension was reduced by 67%, caused by a reduced number of bound cross bridges due to slower cross bridge cycling kinetics. No shift in the force–sarcomere length relationship was observed as SLopt and SL50 were similar. Importantly, the maximal tension of MF was also reduced, by 45%, which was accompanied by a significant reduction of cross bridge cycling kinetics. Thus, despite a largely preserved ultrastructure, muscle fibers of NEM3 patients show significant contractile weakness in the absence of major changes in thin filament length (i.e. unaltered SLopt and SL50). The reduced contractility of single myofibrils indicates that muscle fiber weakness in NEM3 patients is at least partly caused by dysfunctional sarcomeres.

http://dx.doi.org/10.1016/j.nmd.2016.06.176

http://dx.doi.org/10.1016/j.nmd.2016.06.175

P.156 Exome sequencing identifies a novel MYH7 mutation in a patient with nemaline myopathy and cardiomyopathy E. Malfatti 1, X. Lornage 2, A. Behin 3, P. Laforet 3, J. Bohm 2, G. Brochier 3, M. Fardeau 1, J. Laporte 2, N. Romero 1 1 Myology Institute, Paris, France; 2 Dept. of Translational Medicine, IGBMC, Illkirch, France; 3 Centre de référence de Pathologie Neuromusculaire Paris-Est, Institut de Myologie, GHU La Pitié-Salpêtrière, Assistance Publiqu, Paris, France Nemaline myopathies (NM) are rare and severe muscle diseases are characterised by the presence of nemaline bodies (rods) in the muscle biopsy. Eleven genes have been implicated in the aetiology of NM to date. Myosin 7 gene (MYH7), encoding the low/beta-cardiac myosin heavy chain (MHC-b) expressed both in skeletal muscle and the heart, has been found mutated in Laing Distal Myopathy (LDM), Myosin Storage myopathy, (MSM) and Familial Hypertrophic Cardiomyopathy (FHCM). Here we describe a 60-yearold French woman presenting with cardiomyopathy revealed by tachycardic episodes at 32 years, and mild slowly progressive muscular weakness involving pelvic girdle and hamstring muscles from her forties. A deltoid muscle biopsy at 44 years showed the presence of clusters of nemaline bodies in 20% of fibres, multiple and poorly defined irregularities of the intermyofibrillar network, and type 1 fibre disproportion. Whole-body MRI at 55 years confirmed the presence of mild global muscle involvement predominating on spine erectors, gluteus maximus, and thigh muscles. Exome sequencing disclosed a novel

P.157 Contractile dysfunction in permeabilized muscle fibers of NEM6 patients with the Dutch founder mutation in KBTBD13 J. de Winter 1, M. van Willigenburg 1, B. Joureau 1, G. Stienen 1, S. Lassche 2, B. van Engelen 2, N. Voermans 2, C. Ottenheijm 1 1 VU University Medical Center, Amsterdam, Netherlands; 2 Radboud University Medical Centre, Nijmegen, Netherlands Nemaline myopathy (NM) is among the most common non-dystrophic congenital myopathies and is characterized by muscle weakness. Recently, a novel implicated gene was discovered – KBTBD13. The majority of NM patients with KBTBD13 mutations (NEM6) carry the Dutch founder mutation (c.1222C > T, p.Arg408Cys missense mutation). NEM6 patients have muscle weakness, a typical slowness of movement and experience muscle stiffness. Here, we investigated the contractile performance of permeabilized muscle fibers of NEM6 patients to obtain insight into the effect of this mutation on myofilament function. Single muscle fibers were isolated from biopsies of ten NEM6 patients and six control subjects (CTRL). Fibers were permeabilized, mounted in a single fiber setup and activated using exogenous calcium. Tension was determined by dividing force by the fiber’s cross-sectional area. Furthermore, we measured the calcium-sensitivity of force generation, crossbridge cycling kinetics and the passive stiffness of the muscle fiber. Data are expressed as mean ± SEM. The maximal active tension was lower in NEM6 fibers (70 ± 8 mN/mm2 in NEM6 vs. 122 ± 4 mN/mm2 in CTRL). Also, crossbridge cycling kinetics was slower in NEM6 fibers (5.0 ± 0.3 s-1 in NEM6 vs. 8.2 ± 0.9 s-1 in CTRL). Hence, slower cross-bridge cycling kinetics could play a role in the slowness of movement that patients experience. In addition, the calcium-sensitivity of force generation was higher in NEM6 fibers (pCa50: 5.93 ± 0.08 in NEM6 vs. 5.75 ± 0.02 in CTRL), which might contribute to the impaired relaxation kinetics and the muscle stiffness that NEM6 patients experience. No changes were observed in the passive stiffness of NEM6 fibers (46 ± 6 mN/mm2 in NEM6 vs. 49 ± 11 mN/mm2 in CTRL at a sarcomere length of 3.2 μm). Here, we studied the contractile performance of single muscle fibers isolated from biopsies of NEM6 patients. The data suggest that changes at the myofilament level contribute to the clinical phenotype of NEM6. http://dx.doi.org/10.1016/j.nmd.2016.06.177

P.158 Functional studies of YBX3 variants associated with nemaline myopathy L. Sagath 1, J. Laitila 1, V. Lehtokari 1, K. Kiiski 1, M. Grönholm 2, C. Wallgren-Pettersson 1, K. Pelin 2 1 Folkhälsan Institute of Genetics, Helsinki, Finland; 2 University of Helsinki, Helsinki, Finland YBX3 (Y-box binding protein 3, also known as cold shock domain protein A, CSDA) is a protein involved in the regulation of transcription, expressed at high levels in skeletal and heart muscle. It represses myogenin transcription in skeletal muscle and is gradually dephosphorylated and accumulated in the nucleus during muscle differentiation and maturation. This leads to a gradual decrease in myogenin expression. In a Finnish patient with an unusual form of nemaline myopathy, exome sequencing revealed two YBX3 variants, p.Ser34Arg and p.Arg129Trp. The healthy parents were found to carry one variant each, the

Abstracts 2016 / Neuromuscular Disorders 26 (2016) S88–S212 affected child being compound heterozygous. In addition, both the father and the child carry a splice-site variant in intron 1 of TPM3 and a unique missense variant in RYR1. Both variants have been interpreted as being recessive. The missense variants in YBX3 affect highly conserved amino acids in the DNA-binding domain of YBX3. The Ser34 location has been shown to be phosphorylated in vitro, which is thought to be an important mechanism for the formation of nuclear YBX3 complexes and the binding of YBX3 to single-stranded DNA. The highly conserved Arg129 is located close to Ser134, which has been implicated as the phosphorylation/dephosphorylation site regulating the nuclear/cytoplasmic trafficking of YBX3 in myogenic cells. By immunofluorescence imaging, we have confirmed the expression and assessed the localization ofYBX3 in cultured patient myotubes and myoblasts. YBX3 in control myotubes is seen as granulelike formations in the nucleus, while the patient myotubes show a uniform, yet weaker staining of the entire nucleus. The results indicate that transport ofYBX3 into the nucleus and the functional properties of the protein may be impaired by the amino acid substitutions. We will study this further by transfection of wild-type and mutant YBX3 expression vectors into cultured cells, and pinpoint the localization of the proteins within the cells. http://dx.doi.org/10.1016/j.nmd.2016.06.178

P.159 Large copy number variants are common in the nebulin gene K. Pelin 1, V. Lehtokari 2, L. Sagath 2, C. Wallgren-Pettersson 2, K. Kiiski 2 1 University of Helsinki, Helsinki, Finland; 2 The Folkhälsan Institute of Genetics, Helsinki, Finland Approximately 50% of nemaline myopathies (NM) are caused by recessive mutations in the nebulin gene (NEB). In addition to NM, mutations in NEB can cause distal myopathies with or without nemaline rods, and core-rod myopathy. The patients are usually compound heterozygous for two different NEB variants. The size and complexity of NEB with 183 exons spanning 249 kb of genomic sequence make identification of disease-causing variants challenging. The picture is further complicated by a 32 kb triplicate region (TRI) with three identical tandem copies of eight exons and introns in the central part of NEB. Various types of disease-causing variants have been found in NEB: different point mutations affecting conserved splice signals (33%), small indels causing frameshifts (33%), nonsense mutations (22%), missense mutations affecting nebulin-actin or nebulintropomyosin interactions (7%), and large deletions and duplications (5%). Using our custom NM-CGH array, we have detected large pathogenic copy number variants (CNVs) in 12% of 243 NM families studied to date. The size of the CNVs varied from part of one exon (72 bp) to more than half of the gene (133 kb). CNVs affecting the TRI region were identified in 16% of the NM families, and in 5%, the CNV was interpreted to be pathogenic. The normal copy number of NEB TRI is six (three copies per allele). The control sample TRI CNVs deviate by only one copy from the normal six copies, whereas the NM patient samples showed CNVs of up to 4 additional copies in one allele. One patient with the typical form of nemaline myopathy, born to consanguineous parents, had inherited 7 NEB TRI copies from each parent, giving a total of 14 copies. Based on this study, NEB seems to tolerate deviations of one TRI copy, while the addition of two or more copies seems to be pathogenic. NEB CNVs, especially those in the TRI region, are most readily detected by the NM-CGH array.

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Fatigue and muscle weakness are primary symptoms expressed in individuals with RYR1-RM. Oxidative metabolism is a major contributor to skeletal muscle function during aerobic activity. Pre-clinical trials have demonstrated decreased endurance and diminished cell survival in RYR1-RM zebrafish and human myotubes, respectively. There have been no studies exploring local muscle oxygen extraction in individuals with RYR1-RM during exercise. The purpose of this study is to assess local muscle oxygenation response during cycle ergometry. Eleven participants > 18 years of age. Tissue oxygenation Index (TOI) of the vastus lateralis was measured during arterial occlusion of the thigh. Next, the participants performed cycle ergometry to the limit of their exercise tolerance. Gas exchange and NIRS TOI were measured from rest to peak exercise. Participants appeared to have a normal muscle oxygen desaturation pattern (decreased TOI) during arterial occlusion similar to the suggested normal from the literature. Based on gas exchange data (respiratory exchange ratio and oxygen consumption), participants gave maximal effort during cardiopulmonary exercise testing. Interestingly, muscle oxygenation results demonstrated increased TOI suggesting muscle oxygen supply is greater than muscle oxygen consumption at peak exercise. Although participants had a normal pattern of local muscle oxygen desaturation response with occlusion, they did not have a normal systemic muscle function response during exercise. http://dx.doi.org/10.1016/j.nmd.2016.06.180

P.161 Local muscle strength, oxygen extraction capacity, and exercise capacity in adults with RYR1-related myopathies: Exploratory study J. Witherspoon, B. Drinkard, M. Waite, I. Arveson, M. Stockman, M. Jain, K. Meilleur National Institutes of Health, Bethesda, USA Skeletal muscle weakness and fatigue are primary symptoms in patients with RYR1-related myopathies (RYR1-RM). In this patient population, there is skeletal muscle fatty infiltration and oxidative stress affecting muscle function. Near infrared spectroscopy (NIRS) allows for the measurement of maximal oxygen extraction capacity and local muscle desaturation when used with arterial occlusion (AO) and exercise, respectively. The purpose of this study was to determine the influence of local muscle oxidative metabolism and strength on overall exercise capacity. 14 participants > 18 years of age. Maximal desaturation of vastus lateralis was determined using AO with NIRS. Participants performed cycle ergometry to the limit of their exercise tolerance. Two days later, participants performed sustained isometric knee extension at max effort (peak torque) to the point of fatigue. Tissue oxygenation index was recorded using NIRS during both cycle ergometry and sustained isometric exercise. Participants demonstrated an average of 33.8% (+28.0) relative muscle O2 desaturation. Relative O2 desaturation was related to VO2peak (r = .772, p < .01) and anaerobic threshold (r = .629, p < .05). Peak torque was related to peak power (r = .885, p < .01) and VO2peak (r = .844, p < .01) determined during cycle ergometry. Local muscle oxygen extraction capacity and muscle strength may be determinants of exercise capacity in participants with RYR1-RM. http://dx.doi.org/10.1016/j.nmd.2016.06.181

http://dx.doi.org/10.1016/j.nmd.2016.06.179

CONGENITAL MYOPATHIES RYR1 P.160 Skeletal muscle oxygenation in adults with RYR1-related myopathies: Exploratory study J. Witherspoon, B. Drinkard, I. Arveson, M. Stockman, M. Jain, K. Meilleur National Institutes of Health, Bethesda, USA

P.162 Ryanodine-related myopathies: Clinical, histopathologic and genetic heterogeneity among 16 patients from a Portuguese tertiary centre R. Samões, J. Oliveira, R. Taipa, A. Gonçalves, M. Cardoso, T. Coelho, M. Melo-Pires, R. Santos, M. Santos Centro Hospitalar do Porto, Porto, Portugal Mutations in the ryanodine receptor 1 (RYR1) gene are an important cause of congenital myopathy. The phenotype has been expanded beyond the classical autosomal dominant (AD) central core myopathy. Additionally, some RYR1