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 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 [Ca 2+ ] 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.2016.06.175 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-year- old 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 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. http://dx.doi.org/10.1016/j.nmd.2016.06.176 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, cross- bridge 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, cross- bridge 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 proteinA, 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 S134 Abstracts 2016/Neuromuscular Disorders 26 (2016) S88–S212