Ayúdanos a caminar, ayúdanos a vivir. ¡Juntos podemos conseguirlo!
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Ayúdanos a caminar, ayúdanos a vivir. ¡Juntos podemos conseguirlo!
Ayúdanos a caminar, ayúdanos a vivir. ¡Juntos podemos conseguirlo! Paraje de Reolid 2, 30420 Valentín, Murcia, España C.F.G73886483 Email: [email protected] Web: www.conquistandoescalones.org Facebook: www.facebook.com/conquistandoescalones Twitter: @AceDistrofia ÍNDICE • • • ¿Qué es A.C.E.? ........................................................................................................ 4 Resumen y traducción de artículos científicos..........................................................11 Artículos científicos................................................................................................... 23 • Incomplete penetrance in limb-girdle muscular dystrophy type 1F.....................24 Marina Fanin, PhD1, Enrico Peterle, MD1, Chiara Fritegotto, PhD1, Anna C. Nascimbeni, PhD1, Elisabetta Tasca, PhD2, Annalaura Torella, PhD3,4, Vincenzo Nigro, MD, PhD3,4, Corrado Angelini, MD1,2 • Genetic basis of limb-girdle muscular dystrophies: the 2014 update..................26 • P.5.10 - Clinical and ultrastructural changes in transportinopathy......................38 • P.5.12 - A mutation in TNPO3 causes LGMD1F and characteristic nuclear pathology............................................................................................................. 39 Vincenzo Nigro e Marco Savarese C. Angelini 1, E. Peterle 1, M. Fanin 1, G. Cenacchi 2, V. Nigro 3 A. Kubota 1, M.J. Melia 2, S. Ortolano 3, J.J. Vilchez 4, J. Gamez 5, K. Tanji 6, E. Bonilla 6, L. Palenzuela 2, I. Fernandez-Cadenas 2, A. Pristoupilova 7, E. Garcia-Arumi 2, A.L. Andreu 2, C. Navarro 3, R. Marti 2, M. Hirano 1 • Distrofia dei cingoli, Telethon scopre il gene responsabile della rara patologia..40 • Next-Generation Sequencing Identifies Transportin 3 as the Causative Gene for LGMD1F.............................................................................................................. 41 Annalaura Torella1,2, Marina Fanin3, Margherita Mutarelli1, Enrico Peterle3, Francesca Del Vecchio Blanco2, Rossella Rispoli1,4, Marco Savarese1,2, Arcomaria Garofalo2, Giulio Piluso2, Lucia Morandi5, Giulia Ricci6, Gabriele Siciliano6, Corrado Angelini3,7, Vincenzo Nigro1,2 • Clinical phenotype, muscle MRI and muscle pathology of LGMD1F..................48 • Limb-girdle muscular dystrophy 1F is caused by a microdeletion in the transportin 3 gene................................................................................................57 Enrico Peterle1 , Marina Fanin1 , Claudio Semplicini1 , Juan Jesus Vilchez Padilla2 , Vincenzo Nigro3,4 , Corrado Angelini1,5 Maria J. Melià1,2, Akatsuki Kubota3, Saida Ortolano4, Juan J. Vilchez5, Josep Gámez6, Kurenai Tanji7, Eduardo Bonilla3,7,†, Lluis Palenzuela1,2, Israel Fernandez-Cadenas1, Anna Pristoupilová8,9, Elena Garcia-Arumí1,2, Antoni L. Andreu1,2, Carmen Navarro2,4, Michio Hirano3,and Ramon Marti1,2 • P07 Limb-Girdle Muscular Dystrophy and Inherited Myopathy Limb Girdle Muscular dystrophy 1F: Clinical, Molecular and Ultrastructural study (P07.032) ............................................................................................................................. 67 Corrado Angelini1, Enrico Peterle2, Marina Fanin3, Giovanna Cenacchi4 and Vincenzo Nigro5 • Ultrastructural changes in LGMD1F.................................................................... 68 • D.O.3 Next generation sequencing application are ready for genetic diagnosis of muscular dystrophies...........................................................................................73 Giovanna Cenacchi1, Enrico Peterle2, Marina Fanin2, Valentina Papa1, Roberta Salaroli1 and Corrado Angelini2,3 M. Savarese 1, A. Torella 1, M. Mutarelli 2, M. Dionisi 2, T. Giugliano 3, G. Di Fruscio 3, M. Iacomino 3, A. Garofalo 3, S. Aurino 3, F. Del Vecchio Blanco 3, G. Piluso 3, L. Politano 4, M. Fanin 5, C. Angelini 5, V. Nigro 3 2 • New quantitative MRI indexes useful to investigate muscle disease..................74 • Identificazione di nuovi geni coinvolti nelle distrofie muscolari dei cingoli mediante arrays e sequenziamento di nuova generazione (NGS).....................75 C. Angelini, M. Fanin, E. Peterle A. Torella 1, F. Del Vecchio Blanco 3, M. Dionisi 2, A. Garofalo 3, M. Iacomino 3, M. Mutarelli 2 , M. Savarese 1, G. Piluso 1, V. Nigro 1 • LGMD 1(F) - A pathogenetic hypotesis based on histopathology and ultrastructure........................................................................................................ 76 G. Cenacchi, E. Peterle, L. Tarantino, V. Papa, M. Fanin, C. Angelini • A novel autosomal dominant limb-girdle muscular dystrophy (LGMD 1F) maps to 7q32.1-32.2..........................................................................................................77 L.Palenzuela1, PhD; A.L.Andreu1, MD, PhD; J.Gámez2, MD, PhD; M.R.Vilà3, PhD; T.Kumimatsu3, PhD; A.Meseguer1, PhD; C.Cervera2, MD, PhD; I.Fernández Cadenas1, Msc; P.F.M. Van der Ven4, PhD; T.G.Nygaard5, MD; E.Bonilla3, MD; and M. Hirano3, MD • Autosomal dominant limb-girdle muscular dystrophy..........................................80 J. Gamez1, MD; C. Navarro3, MD; A.L. Andreu2, MD; J.M. Fernandez4, MD; L. Palenzuela2, MS; S. Tejeira3, MS; R. Fernandez–Hojas3, MS; S. Schwartz2, MD, PhD; C. Karadimas5, PhD; S. DiMauro5, MD; M. Hirano5, MD; and C. Cervera1, MD 3 ¿Qué es A.C.E.? 4 5 6 7 8 9 10 Resumen y traducción de artículos científicos 11 Diciembre 2014 Carta al editor. Penetrancia Incompleta en la Distrofia Muscular de Cinturas Tipo 1f Marina Fanin, PhD1, Enrico Peterle, MD1, Chiara Fritegotto, PhD1, Anna c. Nascimbeni, PhD1, Elisabetta Tasca, PhD2, Annalaura Torella, PhD3,4, Vincenzo Nigro, MD, PhD3,4, Corrado Angelini, MD1,2 1. Departamento de Neurociencias, Universidad de Padua, Padua, 2. IRCCS Fundación Hospital San Camillo, Venecia, Italia 3. Departamento de Bioquímica, Biofísica y Patología General, II Universidad de Nápoles, Nápoles, Italia 4. Instituto Telethon de Genética y Medicina, Nápoles, Italia La distrofia muscular de cinturas tipo 1F (LGMD) (MIM #608423) es una patología autosómica dominante rara cuyo locus ha sido mapeado y ha sido genéticamente identificado tras la investigación en una extensa familia. Durante el examen de esta familia, ampliamos la genealogía inicial y las características clínicas de la enfermedad. Se caracteriza por un grado variable de debilidad muscular y deterioro funcional, con un inicio de los síntomas o bien antes de los 15 años (forma juvenil) o bien en la década de los 30 a los 40 (forma adulta). La investigación clínicogenética de esta familia reveló que, en algunos pacientes, la enfermedad se transmitió a través de familiares aparentemente no afectados (penetrancia incompleta). Para calcular la tasa de penetrancia exacta, examinamos tanto el fenotipo clínico como el genotipo de 115 miembros de la familia. Nuestros resultados pueden ser útiles para asesoramiento genético, especialmente en los pacientes más jóvenes con la mutación. Mayo 2014 Acta Miológica • 2014; XXXIII: p. 1-12. Bases genéticas de la distrofia muscula de cinturas: actualización de 2014 Vincenzo Nigro y Marco Savarese. Departamento de Bioquímica, Biofísica y Patología General, Segunda Universidad de los Estudios de Nápoles y Telethon Institute of Genetics and Medicine (TIGEM), Nápoles, Italia Las distrofias musculares de cinturas (LGMD) son un grupo altamente heterogéneo de desórdenes musculares, los cuales afectan primero a los músculos voluntarios de la cadera y de los hombros. La definición es altamente descriptiva y menos ambigua por exclusión: no-Xlinked, no-FSH, no-miotónica, no-distal, no sindrómica, y no congénita. Actualmente, la clasificación genética está siendo demasiado compleja, puesto que el acrónimo LGMD ha sido también usado para otros desórdenes miopáticos con fenotipos superpuestos. Hoy en día, la lista de genes por ser analizados es demasiado larga para abordarla gen a gen y sería mejor mirarla con los paneles de Next Generation Sequencing (NGS), que deberían incluir cualquier gen que hasta ahora haya sido asociado con el cuadro clínico de la LGMD. El presente artículo tiene el objetivo de recapitular las bases genéticas de la LGMD ordenando y proponiendo una nomenclatura para las formas huérfanas. Esto es útil dado el ritmo al que salen nuevos descubrimientos. Treinta y un loci han sido ya identificados, ochos autosómicos dominantes y veintitrés autosómicos recesivos. Las formas dominantes (LGMD1) son: LGMD1A (myotilin), LGMD1B (lamin A/C), LGMD1C (caveolin 3), LGMD1D (DNAJB6), LGMD1E (desmin), LGMD1F (transportin 3), LGMD1G (HNRPDL), LGMD1H (chr. 3). The autosomal recessive forms (LGMD2) are: LGMD2A 12 (calpain 3), LGMD2B (dysferlin), LGMD2C (γ sarcoglycan), LGMD2D ( α sarcoglycan), LGMD2E ( β sarcoglycan), LGMD2F (δ sarcoglycan), LGMD2G (telethonin), LGMD2H (TRIM32), LGMD2I (FKRP), LGMD2J (titin), LGMD2K (POMT1), LGMD2L (anoctamin 5), LGMD2M (fukutin), LGMD2N (POMT2), LGMD2O (POMTnG1), LGMD2P (dystroglycan), LGMD2Q (plectin), LGMD2R (desmin), LGMD2S (TRAPPC11), LGMD2T (GMPPB), LGMD2U (ISPD), LGMD2V (Glucosidase, alpha ), LGMD2W (PINCH2). LGMD autosómicas dominantes La LGMD1F fue originalmente mapeada en un intervalo de 3.68 Mb en el cromosoma 7q32.1 7q32.2 en una familia italo-española. Presentamos la identificación del TNPO3 mediante una secuenciación exómica completa de 4 miembros enfermos de la misma familia y el completo perfeccionamiento de la región en el WMS 2012. Los datos fueron entonces publicados: una mutación en la fase de lectura en el gen de la transportina 3 (TNPO3) es compartida por todos los miembros afectados de la familia con un 94% de penetrancia. El gen TNPO3 está compuesta de 23 exones y codifica una proteína de 923 aminoácidos, expresada también en el músculo esquelético. La proteína TNPO3 con la mutación en la fase de lectura es mayor que la de cepa salvaje, dado que le falta el previsto codón de terminación y se encuentra alrededor del núcleo pero no dentro. Los pacientes con un inicio en la adolescencia muestran un fenotipo más severo con una progresión rápida, mientras que los pacientes con un comienzo ya de adultos presentan una progresión más lenta. Presentan una atrofia marcada de los músculos de la cintura pelviana, afectando especialmente el vastus lateralis y el iliopsoas. Es interesante que algunos pacientes presentan disfagia, aracnodactilia e insuficiencia respiratoria. El rango del CK es de 1 -3x. No ha sido encontrada afectación cardíaca. Agradecimientos Este estudio ha sido apoyado principalmente por los fondos de Telethon, Italia (TGM11Z06 to V.N.) y Telethon- UILDM (Unión Italiana de Lucha contra la Distrofia Muscular) (GUP 10006 and GUP11006 to V.N.). Los patrocinadores no han tenido ninguna participación en el estudio, recogida y análisis de los datos, decisión sobre la publicación o preparación del manuscrito. Octubre 2013 P.5.10 - Cambios clínicos y ultraestructurales en la transportinopatía C. Angelini 1, E. Peterle 1, M. Fanin 1, G. Cenacchi 2, V. Nigro 3 1. Universidad de Padua, Italia; 2. Universidad de Bolonia, Bolonia, Italia; 3. TIGEM, Nápoles, Italia; Se han estudiado en 3 biopsias las características musculares histopatológicas, ultraestructurales y genéticas de una amplia familia italo-española con LGMD autosómica dominante, previamente mapeada en 7q32.1-32.2 (LGMD1F). Hemos recopilado los historiales clínicos de 19 pacientes, de un total de 60; en un par de afectados se ha investigado la histopatología de las biopsias musculares (de la madre 1 biopsia, de su hija 2 biopsias consecutivas a los 9 y 22 años de edad). Se ha observado que la edad a la que se manifiesta por primera vez varía de los 2 a los 35 años y lo hace tanto en la cintura pelviana como en la escapular. En 14 casos se ha hallado hipotrofia tanto en los músculos proximales superiores como en las extremidades inferiores, en las pantorrillas. La gravedad no ha aumentado en las siguientes generaciones. Conclusiones clínicas no notificadas precedentemente son aracnodactilia, disfagia y disartria. Por otra parte, hemos encontrado discrepancia entre la severidad clínica y la biopsia muscular: la hija tiene un pronóstico clínico más grave, en la primera biopsia tenía únicamente atrofia de las fibras tipo 1, mientras que la atrofia de las fibras ha aumentado en la segunda biopsia. La madre 13 tenía una histopatología del músculo más severa (más variación de las fibras musculares y cambios autofágicos con tinción de fosfatasa ácida). La causa de la atrofia progresiva y de la pérdida de miofibrillas es un ensamblaje sarcomérico anormal. Mediante microscopio electrónico ha sido revelada una acumulación de cuerpos miofibrilares en las fibras musculares. Se ha observado una acumulación de desmina y miotilina y agregados p62. Se ha descubierto como causa de esta enfermedad un defecto en el gen de la transportina 3 que representa un nuevo mecanismo de miopatía dominante. Nuestros datos morfológicos y utraestructurales parecen seguir un fenotipo similar a las enfermedades miofibrilares; sin embargo, había presencia de autofagosomas también. Es posible que las proteínas SR no puedan migrar o ser transportadas fuera o dentro de la membrana nuclear. Octubre 2013 P.5.12 - Una mutación en TNPO3 causa LGMD1F y una patología nuclear característica A. Kubota 1, M.J. Melia 2, S. Ortolano 3, J.J. Vilchez 4, J. Gamez 5, K. Tanji 6, E. Bonilla 6, L. Palenzuela 2, I. Fernandez-Cadenas 2, A. Pristoupilova 7, E. Garcia-Arumi 2, A.L. Andreu 2, C. Navarro 3, R. Marti 2, M. Hirano 1 1. Centro Médico de la Universidad de Columbia, Departamento de Neurología, Nueva York, Estados Unidos; 2. Vall d'Hebron Instituto de Investigación, Universidad Autónoma de Barcelona, Grupo de Investigación de Desórdenes Neuromusculares y Mitocondriales, Barcelona, España; 3. Instituto de Investigación Biomédica de Vigo, Hospital Universitario de Vigo, Departamento de Patología y Neuropatología, Vigo, España 4. Hospital Universitario y Politécnico La Fe, Departamento de Neurología, Valencia, España; 5. Hospital Universitario Vall d'Hebron, Instituto de Investigación, Universidad Autónoma de Barcelona, Clínica de Desórdenes Neuromusculares, Departamento de Neurología, Barcelona, España; 6. Centro Médico de la Universidad de Columbia, Departamento de Patología y Biología Celular, Nueva York, Estados Unidos; 7. Centro Nacional de Análisis Genómico, Barcelona, España La distrofia Muscular de Cinturas 1F (LGMD1F) es una patología autosómica dominante que afecta a una familia española. Mediante una secuenciación genómica completa, se ha identificado la supresión de un único nucleótido (c.2771del) en el gen de la transportina 3 en un paciente con LGMD1F. La mutación interrumpe el codón de terminación de la TNPO3 y causa una mutación en la fase de lectura. La transportina 3 es una proteína nuclear y media la importación de las proteínas ricas en serina-arginina al núcleo, que son importantes para el splicing del mRNA. El objeto de estudio es el análisis de la transportina 3 en la patogénesis de la LGMD1F. Se ha realizado una secuenciación del TNPO3 mediante didesoxi en 24 pacientes afectados y 23 familiares sanos. Las muestras de tejido muscular de 4 pacientes han sido analizadas mediante métodos convencionales e inmunohistoquímica. Una secuenciación directa de la TNPO3 ha mostrado que todos los pacientes presentaban una mutación heterocigota y ninguno de los familiares sanos tenía la mutación. Las tinciones del músculo con hematoxilina-eosina (HE) han revelado núcleos (10.7 ± 3.0%; media ± SD) con palidez central en todos los pacientes estudiados. La inmunohistoquímica con anticuerpos antritransportina 3 muestran una co-localización con los núcleos en los sujetos de control. En los pacientes, se ha observado también la transportina 3 en el núcleo, pero a menudo también distribuida en la periferia, en un patrón de coloración a manchas similar al observado con HE. Los estudios genéticos e histológicos en una familia española sostienen fuertemente la hipótesis de que el gen TNPO3 es la causa genética de la LGMD1F. Los estudios patológicos indican también que la distribución subcelular de la transportina 3 es interrumpida y daña la estructura de los núcleos. 14 Mayo 2013 Secuenciación de última generación identifica la Transportina 3 como causa genética de la LGMD1F Annalaura Torella1,2, Marina Fanin3, Margherita Mutarelli1, Enrico Peterle3, Francesca Del Vecchio Blanco2, Rossella Rispoli1,4, Marco Savarese1,2, Arcomaria Garofalo2, Giulio Piluso2, Lucia Morandi5, Giulia Ricci6, Gabriele Siciliano6, Corrado Angelini3,7, Vincenzo Nigro1,2 1. TIGEM (Telethon Institute of Genetics and Medicine), Nápoles, Italia 2. Departamento de Bioquímica, Biofísica y Patología General, Segunda Universidad de los Estudios de Nápoles, Italia 3. Departamento de Neurociencias, Universidad de los Estudios de Padua, Italia 4. Investigación del Cáncer UK, Londres, Reino Unido 5. Fundación IRCSS Instituto Neurológico C. Besta, Milán, Italia 6. Departamento de Medicina Clínica y Experimental, Universidad de los Estudios de Pisa, Pisa, Italia 7. IRCSS S. Camillo, Venecia, Italia Resumen Las Distrofia Musculares de Cinturas (LGMD) tienen condiciones genéticas y clínicas heterogéneas. Hemos estudiado una familia con un patrón de transmisión autosómica dominante, previamente clasificada como LGMD1F y mapeada en el cromosoma 7q32. Los miembros afectados se caracterizan por debilidad muscular que daña primero la cintura pelviana y el iliopsoas. Hemos secuenciado el exoma completo de 4 miembros de la misma familia y hemos identificado una variante compartida de la mutación heterocigota de la fase de lectura en el gen transportina 3 (TNPO3), que modifica un miembro de la superfamilia importina- β. El gen TNPO3 ha sido mapeado en el intervalo crítico de la LGMD1F y el producto génico humano de sus 923 aminoácidos es expresado también en el músculo esquelético. A parte, hemos identificado un caso aislado de LGMD con una nueva mutación "sin sentido" en el mismo gen. Hemos localizado el TNPO3 mutante entorno al núcleo, pero no dentro. La implicación genética conectada con el transporte al núcleo sugiere un nuevo mecanismo patológico que conduce a la distrofia muscular. Abril 2013 Fenotipo clínico, MRI muscular y patología muscular de la LGMD1F Enrico Peterle • Marina Fanin • Claudio Semplicini • Juan Jesus Vilchez Padilla • Vincenzo Nigro • Corrado Angelini 1. Departamento de Neurociencias, Universidad de Padua, Campus Biomedico “Pietro d’Abano”, Padua Italia 2. Departamento de Neurología, Hospital Universitario y Politécnico de La Fe, Valencia, España 3. Segunda Universidad de los Estudios de Nápoles, Departamento de Patología General, Nápoles, Italia; 4. TIGEM (Telethon Institute of Genetics and Medicine), Nápoles, Italia 5. IRCSS S. Camillo, Venecia, Italia Resumen De las 7 diversas formas genéticas autosómicas dominantes de LGMD descritas hasta hoy, solamente en 4 ha sido identificado el gen que la causa (LGMD1A-1D). Describimos las características clínicas, histopatológicas y MRI musculares de una familia italo-española con 15 LGMD1F que presenta debilidad en los músculos proximales de las extremidades y en los músculos axiales. Hemos obtenido datos clínicos completos y clasificado la progresión de la enfermedad en 29 pacientes. Se ha realizado una MRI muscular en 7 pacientes. Se han estudiado 3 biopsias musculares de 2 pacientes. Los enfermos con una edad de manifestación en las primeras décadas presentan un fenotipo más severo con una progresión rápida de la enfermedad, aquellos con una manifestación ya de adultos presentan un desarrollo más lento. La MRI muscular muestra una importante atrofia en los músculos de las extremidades inferiores, especialmente en el vastus lateralis. Ampliar la población de pacientes ha permitido la identificación de características previamente no detectadas, incluyendo disfagia, aracnodactilia e insuficiencia respiratoria. Las biopsias musculares muestran atrofia difusa de las fibras que evoluciona con el tiempo, cambios miopáticos crónicos, áreas basófilas citoplasmáticas, autofagosomas y agregados miofibrilares y proteínas citoesqueléticas. La LGMD1F se caracteriza por una afectación selectiva de los músculos de las extremidades e insuficiencia respiratoria en fase avanzada y en diversos grados de progresión clínica. Nuevas características clínicas han emergido del estudio de posteriores pacientes. El empleo de secuenciadores de última generación (NGS) en esta familia ha dado como resultado la reciente identificación de la transportina 3 (TNPO3) como la causa genética de la LGMD1F. Estos nuevos resultados son cruciales para entender el nexo entre los mecanismos patogenéticos y las características clínicas. Marzo 2013 La Distrofia Muscular de Cinturas 1F causada por una microdeleción en el gen Transportina 3. Maria J. Melià1,2, Akatsuki Kubota3, Saida Ortolano4, Juan J. Vilchez5, Josep Gámez6, Kurenai Tanji7, Eduardo Bonilla3,7,†, Lluis Palenzuela1,2, Israel Fernandez-Cadenas1, Anna Pristoupilová8,9, Elena Garcia-Arumí1,2, Antoni L. Andreu1,2, Carmen Navarro2,4, Michio Hirano3,and Ramon Marti1,2 1. Grupo de Investigación de Desórdenes Neuromusculares y Mitocondriales, Vall d’Hebron Instituto de Investigación, Universidad Autónoma de Barcelona, Barcelona,08035, España 2. Centro de Investigación Biomédica en Red Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Madrid, 28029, España 3. Departamento de Neurología, Centro Médico de la Universidad de Columbia, Nueva York, NY 10032, USA 4. Departamento de Patología y Neuropatología, Instituto de Investigación Biomédica de Vigo, Hospital Universitario de Vigo, Vigo, 36200, España 5. Departamento de Neurología, Hospital Universitario y Politécnico de La Fe, Valencia, 46026, España, y Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas, Instituto de Salud Carlos III, Madrid, 28029, España 6. Clínica de Desórdenes Neuromusculares, Departamento de Neurología, Hospital Universitario Vall d'Hebron, Instituto de Investigación, Universidad Autónoma de Barcelona, Barcelona, 08035, España 7. Departamento de Patología y Biología Celular, Centro Médico de la Universidad de Columbia, Nueva York, NY 10032, USA 8. Centro Nacional de Análisis Genómico, Barcelona, 08028, España 9. Instituto de Desórdenes Metabólicos Hereditarios, Primera Facultad de Medicina, Universidad Charles en Praga, 12808, República Checa En 2001, hemos detectado el vínculo genético de una forma autosómica dominante de distrofia muscular de cinturas, la distrofia muscular de cinturas 1F, en el cromosoma 7q32.1-32.2, pero la identificación del gen mutante era elusiva. Ahora, usando una estrategia de secuenciación del genoma entero, hemos identificado la causa de la mutación de la distrofia de cinturas 1F, una deleción heterocigota de un solo nucleótido (c.2771del) en el codón de terminación de la 16 Transportina 3 (TNPO3). Este gen se coloca dentro de la región cromosómica conectada a la enfermedad y codifica una proteína de la membrana nuclear perteneciente a la familia de las betaimportinas. La TNPO3 transporta proteínas ricas en serina/arginina al núcleo y ha sido identificada como un factor clave en el proceso de importación del VIH al núcleo. La mutación genera una extensión de 15 aminoácidos en la terminación C de la proteína, se aisla con el fenotipo clínico y está ausente en la base de datos de la secuencia genómica y en un grupo de >200 alelos de control. En el músculo esquelético de los individuos afectados, la expresión del ARN mensajero mutante y las anomalías histológicas de los núcleos y de la TNPO3 indican una función alterada del TNPO3. Nuestros resultados demuestran que la mutación del TNPO3 es la causa de la distrofia muscular de cinturas 1F, amplían nuestro conocimiento de las bases moleculares de las distrofias musculares y refuerzan la importancia de los defectos de las proteínas de la envoltura nuclear como causa de miopatías hereditarias. Febrero 2013 P07 - Distrofias musculares de cinturas y miopatías hereditarias. Distrofia muscular de cinturas 1F: estudio de las alteraciones clínicas, moleculares y ultraestructurales. (P07.032) Corrado Angelini1, Enrico Peterle2, Marina Fanin3, Giovanna Cenacchi4 and Vincenzo Nigro5 1. 2. 3. 4. 5. Universidad de Neurociencias de Padua, Padua, PD, Italia Universidad de Neurociencias de Padua, Padua, PD, Italia Universidad de Neurociencias de Padua, Padua, PD, Italia Universidad de Patología de Bolonia, Bolonia, BO, Italia Universidad de Patología General de Nápoles, Nápoles, NA, Italia CONTEXTO: Las LGMD son un grupo heterogéneo de enfermedades genéticas con debilidad en los músculos proximales de las extremidades y/o los distales. Hasta hoy se han descubierto 8 formas de LGMD autosómicas dominantes. El fenotipo clínico de la LGMD1F está caracterizado por una notable variabilidad, que va de una manifestación precoz, con una progresión rápida y severa a formas menos agresivas. Las características clínicas y morfológicas de los pacientes con LGMD1F no han sido aun suficientemente caracterizadas como para sugerir una etiología específica. MÉTODO: Hemos recopilado los datos clínicos de 19 de 60 pacientes y hemos ampliado el árbol genealógico; en un par de afectados (madre 1 biopsia, su hiha 2 biopsias consecutivas a los 9 y 22 años) ha sido estudiado el análisis histopatológico, la inmunohistoquímica (desmina, miotilina, p62) y microscopía electrónica de las biopsias musculares. El ADN de 4 pacientes ha sido estudiado con la plataforma MotorChip CGH array para identificar el gen responsable. RESULTADO: Se ha observado que la edad de manifestación variaba de los 2 a los 35 años; en la mitad de los casos se ha hallado hipotrofia tanto en los músculos proximales superiores como en las extremidades inferiores, en las pantorrillas. A parte, hemos hallado discrepancia entre la gravedad clínica y la implicación de la biopsia muscular: la hija (caso de referencia) tiene una progresión clínica más grave, y una mayor atrofia de las fibras musculares, en vez la madre tiene una histopatología del músculo más comprometida (más variabilidad de las fibras musculares y cambios autofágicos con tinción de fosfatasa ácida). Se ha observado una acumulación de desmina y miotilina y agregados p62. Mediante un microscopio electrónico se ha identificado una acumulación de cuerpos miofibrilares en las fibras musculares. La MRI muscular en los pacientes de referencia muestra una severa y selectiva atrofia en el vastus lateralis. CONCLUSIONES: Nuestros estudios morfológicos y ultraestructurales parecen sugerir una miopatía con fenotipo análogo a aquellos descritos por enfermedades Z-disk. Aunque el defecto genético específico no ha sido todavía encontrado, es posible hacer la hipótesis de que la 17 LGMD1F pueda llevar una descomposición de la red citoesquelética correlacionada con la desmina. Con el apoyo de: Telethon Itali, AFM (Association Francaise contre les Myopathies). Declaraciones: el Dr. Angelini ha recibido una compensación personal por las actividades con Genzyme como miembro de Comité Consultivo. El Dr. Peterle no tiene nada que comunicar. La Dra. Fanin no tiene nada que comunicar. La Dra. Cenacchi no tiene nada que comunicar. El Dr. Nigro no tiene nada que comunicar. Diciembre 2012 Alteraciones ultraestructurales en la LGMD1F Giovanna Cenacchi1, Enrico Peterle2, Marina Fanin2, Valentina Papa1, Roberta Salaroli1 and Corrado Angelini2,3 1. Departamento de Ciencias Biomédicas y Neuromotoras, "Alma Mater" Universidad de Bolonia, Bolonia 2. Departamento de Neurociencias, Universidad de Padua, Padua 3. IRCCS S.Camillo, Venecia, Italia En una amplia familia italo-española con herencia autosómica dominante se ha hallada debilidad muscular de los músculos proximales de las extremidades y de los músculos axiales. Han sido descritas las características clínicas, genéticas e histológicas. Ha sido previamente identificado el locus en el cromosoma 7q32.1-32.2 para esta distrofia muscular de cinturas 1F (LGMD1F). Hicimos informe de un estudio patológico muscular de 2 pacientes (madre e hija), de esta familia. Los resultados morfológicos musculares muestran un incremento de la variabilidad de las dimensiones de las fibras, atrofia de las fibras y vacuolas positivos a la fosfatasa ácida. La inmunofluorescencia por desmina, miotilina, p62 y LC3 ha mostrado una acumulación de miofibrillas, agregados de proteínas aglutinantes de la ubiquitina y autofagosomas. El estudio ultraestructural confirma los vacuolas autofagosomales. Han sido detectadas diversas alteraciones de los componentes miofibrilares, como un desorden importante, estructuras de varilla con aspecto granular y ocasionalmente cuerpos citoplasmáticos. Nuestros datos ultraestructurales y las características patológicas musculares son características de la LGMD1F y sostienen la hipótesis de que los defectos genéticos llevan a una miopatía fenotípica asociada a una descomposición de la red citoesquelética. Nuestros datos morfológicos y ultraestructurales sugieren en nuestros casos de LGMD1F una miopatía fenotípica similar a aquellas descritas por las enfermedades Z-disk. Aunque los defectos genéticos están aún en fase de estudio, es posible hacer la hipótesis de que la proteína mutante en LGMD1F pueda provocar una descomposición de la red citoesquelética relacionada con la desmina. Agosto 2012 D.O.3 - Las aplicaciones de la secuenciación de última generación están listas para diagnóstico genético de distrofias musculares M. Savarese 1, A. Torella 1, M. Mutarelli 2, M. Dionisi 2, T. Giugliano 3, G. Di Fruscio 3, M. Iacomino 3 , A. Garofalo 3, S. Aurino 3, F. Del Vecchio Blanco 3, G. Piluso 3, L. Politano 4, M. Fanin 5, C. Angelini 5, V. Nigro 3 18 1. Segunda Universidad de los Estudios de Nápoles, Laboratorio de Genética Médica, Departamento de Patología General, Nápoles, Italia; 2. TIGEM, Telethon Institute of Genetic and Medicine, Nápoles, Italia; 3. Segunda Universidad de los Estudios de Nápoles, Departamento de Patología General, Nápoles, Italia; 4. Segunda Universidad de los Estudios de Nápoles, Cardiomiología y Genética Médica, Nápoles, Italia; 5. Universidad de los Estudios de Padua, Departamento de Neurociencias, Padua, Italia La secuenciación de última generación (NGS) está causando un fuerte impacto en nuestro conocimiento de los diversos aspectos de la biología. Puede ser por otra parte muy potente para estudiar pacientes con condiciones genéticas heterogéneas, como las distrofias musculares. En primer lugar para identificar nuevos genes usando la resecuenciación del exoma. En segundo lugar, para diagnosticar mutaciones en todos los genes causantes conocidos, si se utilizan con enfoque específico. En tercer lugar, para obtener conocimiento sobre el impacto de las mutaciones en la expresión y splicing del mRNA en los músculos afectados. Hemos utilizados NGS para identificar nuevos genes a través de la secuenciación del entero cromosoma. Hemos secuenciado el exoma entero de 4 miembros de la familia con LGMD1F separados por más de once meiosis y ha sido identificada una única nueva variante frame shift heterocigota compartida. Esto causa una alteración sin fin en el gen de la Transportina 3 (TNPO3) que codifica un miembro de la superfamilia de las importinas-beta. Para realizar la segunda tarea, hemos recopilado 160 casos de familiares con distrofia muscular de cinturas no específica con una aparente herencia autosómica. Todas las muestras de ADN han sido primero enriquecidas con 486,480 bp, con una cobertura de 2447 exones de 98 genes usando la tecnología Haloplex con el uso de códigos de barras. Hemos realizado agregados NGS de todas las muestras e identificado un número de mutaciones, verificadas después con secuenciación Sanger. Los casos han sido también estudiados con la plataforma AgilentMotorChip CGH array versión 3.0 para identificar deleciones o duplicaciones. Finalmente, en los casos seleccionados, hemos realizado RNA-Seq partiendo de una muestra de biopsia muscular. Hemos convertido mRNA en cDNA y lo hemos purificado con un personalizado SureSelec t Target Enrichment System, focalizado sobre los mismos 98 mRNAs. Las sondas tienen una cobertura 4x con un target total de 1.41 Mb de secuencias/muestras. Estos cDNA han sido secuenciados usando códigos de barras tratando de obtener una cobertura de secuenciación de 100x. Nuestros resultados confirman que hay una heterogeneidad muy alta en las distrofias musculares y que los test de DNA y RNA basados en NGS están listos para uso diagnóstico. Junio 2012 Nuevos índices cuantitativos de MRI útiles para el estudio de enfermedades musculares C. Angelini, M. Fanin, E. Peterle (Padova, IT) CONTEXTO: Proponemos nuevos modelos de medida cuantitativa de la atrofia muscular: el índice del cuádriceps (QI) y el índice del vastus lateralis izquierdo (VLI) midiendo su área mediante MRI. MÉTODO: Hemos usado secuencias T1 de la MRI del músculo del muslo, a alrededor de 15 cm de la cabeza del fémur (segunda slide de la MRI en las extremidades inferiores). En estas secuencias hemos medido el área muscular del cuádriceps femoral izquierdo y del vastus lateralis izquierdo. Estas mediciones han sido realizadas en 11 pacientes con diversos tipos de miopatía p.e. dos casos de miopatías de acumulación de lípidos, una esclerosis lateral amiotrófica, 1 distrofia facio-escápulo-humeral, 1 miopatía miofibrilar, 1 miopatía metabólica, 2 pacientes con LGMD2A, 1 paciente con LGMD1F, 1 miositis osificante, 1 miopatía inespecífica. Las biopsias 19 musculares de estos pacientes han sido también analizadas con morfometría y marcadores moleculares de la atrofia o autofagia, p.e. MURF, LC3. RESULTADOS: Hemos realizado las mediciones del área muscular del cuádriceps femoral (QI) en 11 pacientes, que ha resultado ser de media de 3711 mm 2 ± SD 792. EN este grupo de pacientes hemos identificado 2 subgrupos, uno que incluye 5 pacientes con un alto grado de atrofia muscular (grupo con alta atrofia) cuyos valores están comprendidos entre los 2400 y los 3400 mm 2 (media 2966) y uno que incluye 6 pacientes con un bajo grado de atrofia (grupo con baja atrofia), cuyos valores están comprendidos entre los 3700 y los 5000 mm2 (media 4332). Las mediciones del área muscular del vastus lateralis en 11 pacientes daban de media unos 963 mm2 ± 303.En el subgrupo atrófico el valor estaba comprendido entre los 400 y los 900 mm 2 (media 658,7), mientras en el subgrupo normal el valor estaba comprendido entre los 900 y los 1400 mm2 (media 1217,8). CONCLUSIONES: Sea el índice de los cuádriceps que el del vastus lateralis parece útil para valorar la atrofia muscular en las LGMD, SLA y miopatías metabólicas: un alto grado de atrofia del QI ha sido hallado en las calpainopatías, enfermedades de la motoneurona y distrofia muscular de cinturas del tipo 1F, la medición del VLM ha sido menos específica dado que comprende una área más basta. Ambos índices cuantitativos obtenidos de la MRI muscular pueden ser usados como resultados clínicos de la terapia en enfermedades neuromusculares para seguir y estudiar la historia natural o los efectos de los varios tipos de terapia (esteroides, carnitina, etc.). Un prometedor campo de investigación parece ser la correlación de los índices de las imágines con otros parámetros de atrofia obtenidos en las biopsias musculares, p.e. con sección o fibras o marcadores moleculares de la atrofia y autofagia. Octubre 2011 Actas de la XI Conferencia de la Asociación Italiana de Miología Cagliari, Mayo de 2011 LGMD1F - Una hipótesis patogénica basada en la histopatología y utraestructura G. Cenacchi, E. Peterle, L. Tarantino, V. Papa, M. Fanin, C. Angelini Departamento clínico de las Ciencias Radiológicas e Histopatológicas, Universidad de Bolonia Departamento de Neurociencas y VIMMM, Universidad de Padua En una amplia familia italo-española con aparente herencia autosómica dominate ha sido detectada debilidad muscular de los músculos proximales de las extremidades y de los músculos axiales. Han sido descritas las características clínicas, genéticas e histológicas en 5/32 pacientes. Ha sido previamente identificado el locus en el cromosoma 7q32.1-32.2, pero ningún defecto ha sido detectado en la Filamina C, un gen candidato de esta región cromosómica que codifica la proteína de unión a la actina altamente expresada en el músculo. Hemos hecho informe de un estudio clínico-patológico de dos pacientes (madre e hija) de la misma familia española. La edad de manifestación ha sido en la adolescencia: una manifestación más precoz en la hija con una debilidad más precoz confirma una aparente anticipación genética. Los resultados morfológicos han sido similares en ambos casos: H&E detecta una aumentada variabilidad de la dimensión de las fibras, atrofia de las fibras, tejido conectivo endo y perimisial y vacuolas positivas a la fosfatasa ácida. El estudio ultraestructural confirma atrofia de las fibras, agregados mitocondriales anormales y vacuolas autofagosomales que contienen detritos celulares e imágenes de pseudo mielina: no han sido halladas inclusiones filamentosas que están normalmente asociadas a HIBM (miopatía hereditaria de cuerpos de inclusión). Muchas alteraciones de componentes miofibrilares 20 han sido fácilmente detectadas así como un importante desorden, estructuras de varillas con aspecto granular y ocasionalmente cuerpos citoplasmáticos. Nuestros datos morfológicos sostienen la hipótesis de que otras proteínas codificantes de la actina como la FSCN3 y la KIAA0265 de la misma región crítica, pueden representar interesantes genes candidatos en el mecanismo patogenético en la LGMD1F. Agosto 2003 Una nueva distrofia muscular de cinturas autosómica dominante (LGMD1F) mapeada en el 7q32.1-32.2 L.Palenzuela, PhD; A.L.Andreu, MD, PhD; J.Gámez, MD, PhD; M.R.Vilà, PhD; T.Kumimatsu, PhD; A.Meseguer, PhD; C.Cervera, MD, PhD; I.Fernández Cadenas, Msc; P.F.M. Van der Ven, PhD; T.G.Nygaard, MD; E.Bonilla, MD; and M. Hirano, MD 1. Centre d’Investigacions en Bioquímica i Biologia Molecular (CIBBIM) Hospital Universitario Vall d’Hebron, Barcelona, España; 2. Servei de Neurologia, Hospital Universitario Vall d’Hebron, Barcelona, España; 3. Departamento de Neurología, Columbia University College of Physicians and Surgeons, New York, USA; 4. Departamento de Biología Celular, Universidad de Potsdam, Alemania; 5. Departamento de Neurología, University of Medicine and Dentistry New Jersey Medical School, Newark, NJ. RESUMEN: EN el 2001, los autores describen las características clínicas de una distrofia muscular de cinturas autosómica dominante (LGMD1F) genéticamente distinta. Examinando el entero genoma con más de 400 marcadores microsatélites, los autores han identificado una nueva enfermedad LGMD cuyo locus está en el cromosoma 7q32.1-32.2. En el interior de esta región cromosómica, la Filamina C, un gen que codifica proteínas unificadoras de la actina altamente expresadas en el músculo, era un obvio gen candidato, sin embargo los autores no han detectado ningún defecto en la Filamina C o su producto proteico. Las distrofias musculares de cinturas (LGMD) comprenden un grupo heterogéneo de enfermedades hereditarias caracterizadas por una progresiva y predominante debilidad de los músculos proximales con signos histológicos de necrosis y regeneración en el músculo. A día de hoy, han sido identificadas 15 formas genéticamente diversas de LGMD. Hace dos años, describimos las características clínicas, histológicas y genéticas de una amplia familia española de más de 5 generaciones con LGMD y aparente herencia autosómica dominante (AD); 44 de 76 (58%) hijos de padres afectados manifiestan la enfermedad. El examen clínico de 61 personas ha demostrado una debilidad muscular progresiva en 32 de ellas, afectando principalmente los músculos de cinturas pelviana y escapular. El análisis de vínculo genético molecular para examinar los locis cromosómicos asociados a otras formas de LGMD autosómicas dominantes han demostrado que esta parentela tiene una forma genética diferente de LGMD-AD. Para localizar el locus cromosómico de la enfermedad, hemos emprendido un escaneo de todo el genoma usando marcadores microsatélite. 21 Agosto 2000 Distrofia muscular de cinturas autosómica dominante. Una amplia parentela con signos de anticipación J. Gamez, MD; C. Navarro, MD; A.L. Andreu, MD; J.M. Fernandez, MD; L. Palenzuela, MS; S. Tejeira, MS; R. Fernandez–Hojas, MS; S. Schwartz, MD, PhD; C. Karadimas, PhD; S. DiMauro, MD; M. Hirano, MD; and C. Cervera, MD 1. Departamento de Neurología, Hospital Universitario Vall d’Hebron, Barcelona, España; 2. Centre d’Investigacions en Bioquímica i Biologia Molecular (CIBBIM) Hospital Universitario Vall d’Hebron, Barcelona, España; 3. Departamento de Patología e Neuropatología, Hospital de Meixoeiro; 4. Departamento de Neurofisiología Clinica, Hospital Xeral-Cies, Vigo, España; 5. H. Houston Merritt Clinical Research Center for Muscular Dystrophy and Related Diseases, Departamento de Neurología, Columbia University College of Physicians and Surgeons, New York; RESUMEN: han sido identificadas 14 formas de distrofia muscular de cinturas (LGMD) genéticamente difernetes, incluidos 5 tipos autosómicos dominante (LGMD-AD). OBJETIVO: Describir las características clínicas, histológicas y genéticas de una vasta familia con LGMD y aparente herencia autosómica dominante de más de 5 generaciones. METODO: los autores han examinado 61 miembros de la familia; han sido realizadas biopsias musculares a 5 pacientes. El análisis del linkage ha valorado los loci cromosómicos asociados a otras formas de LGMD-AD. RESULTADOS: Un total de 32 individuos presentan debilidad de las cinturas escapular y pelviana. La severidad parece empeorar en las sucesivas generaciones. Los resultados de la biopsia muscular han sido no específicos y compatibles con distrofia muscular. El análisis del linkage con los cromosomas 5q31, 1q11-q21, 3p25, 6q23, y 7q, ha demostrado que esta enfermedad no es alélica en las LGMD tipo 1A, 1B, 1C, 1D y 1E. CONCLUSIONES: esta familia tiene una forma genéticamente diferente de LGMD-AD. Los autores están de momento realizando un escaneo del genoma entero para identificar el locus de la enfermedad. 22 Artículos científicos 23 December 9th, 2014 LETTER TO THE EDITOR INCOMPLETE PENETRANCE IN LIMB-GIRDLE MUSCULAR DYSTROPHY TYPE 1F Limb-girdle muscular dystrophy (LGMD) type 1F (MIM # 608423) is a rare autosomal dominant disorder whose locus was mapped1,2 and gene identified3,4 by investigating the same large family. During examination of this family, we expanded the original pedigree and the clinical features of the disease. It is characterized by a variable degree of muscle weakness and functional impairment, with onset of symptoms either before age 15 (juvenile form) or in the third to fourth decades (adult form).5 The clinical2genetic investigation of this family revealed that, in some patients, the disease was transmitted through apparently unaffected parents (incomplete penetrance). To calculate the exact penetrance rate, we examined both the clinical phenotype and the genotype of 115 family members. The attribution of clinical status (either affected or unaffected) was based on a neuromuscular examination performed by the same physician using a standardized protocol and by a questionnaire that we designed to identify main disease symptoms (i.e., muscle weakness, gait difficulty, and dysphagia). One hundred fifteen individuals (including the 27 subjects investigated in the original search for the gene defect in this family3 and 88 new individuals previously untested) underwent DNA sample collection (obtained after written consent). The genotype (either mutant or non-mutant) was defined using a mutation-specific test [amplification refractory mutation system2polymerase chain reaction (ARMS-PCR)] and confirmed by DNA sequencing. The mutation segregating in this family (c.2771delA in TPNO3 gene encoding transportin-33) was identified in 45 of 115 individuals (39%) (Fig. 1); among 45 mutant cases, 39 (86.7%) were affected (at a mean age of 47.5 years) and 6 (13.3%) were unaffected. Two unaffected subjects were younger than age 15 years, with a future chance of developing the disease, and 4 were adult “non-penetrant” individuals (at a mean age of 31.5 years). Furthermore, 3 additional unaffected adults whose DNA was unavailable, were obligate carriers of the disease based on the pattern of inheritance. Overall, the penetrance rate was estimated to be 84.7%. C 2014 Wiley Periodicals, Inc. V Letter to the Editor We observed that clinical signs and symptoms of the disease were progressively more likely to manifest with increasing age (Fig. 1). This indicates that, in LGMD1F, FIGURE 1. (A) Mutation-specific test (ARMS-PCR) showing that the wild-type allele generates a 195 bp-sized band (wt) and that the mutant allele generates a 221-bp band (m). Mutant patients (m) display a doublet of bands corresponding to the presence of both heterozygous mutant and wild-type alleles. (B) Electropherograms showing DNA sequence in a control (wild-type) and a mutant patient. The position of the single nucleotide deletion causing a non-stop mutation is indicated by the arrow. (C) Histogram showing age-dependent penetrance rate in LGMD1F: the proportion of affected patients among mutant cases progressively increases with the age of individuals (numbers in parentheses indicate number of individuals in each age group). MUSCLE & NERVE Month 2015 1 24 December 9th, 2014 the penetrance is age-dependent. Incomplete penetrance may be the effect of modifier genes or may due to environmental factors. Although the contribution of epigenetic factors was not explored in this study, we did investigate the potential role of lifestyle and associated conditions in determining disease manifestations. These data show that age-related penetrance is a characteristic feature of LGMD1F that reduces the predictive value of the genetic test. Because age-related penetrance is a major challenge when attempting to quantify the genetic risk of a patient’s offspring, our results may be useful for genetic counseling, especially in younger patients with the mutation. Marina Fanin, PhD1 Enrico Peterle, MD1 Chiara Fritegotto, PhD1 Anna C. Nascimbeni, PhD1 Elisabetta Tasca, PhD2 Annalaura Torella, PhD3,4 3,4 1 Department of Neurosciences, University of Padova, Padova, Italy 2 IRCCS Fondazione San Camillo Hospital, Venice, Italy 3 Department of Biochemistry, Biophysics and General Pathology, II University of Naples, Naples, Italy 4 Telethon Institute of Genetics and Medicine, Naples, Italy 1. Gamez J, Navarro C, Andreu AL, Fernandez JM, Palenzuela L, Tejeira S, et al. Autosomal dominant limb-girdle muscular dystrophy: a large kindred with evidence for anticipation. Neurology 2001;56:450–454. 2. Palenzuela L, Andreu AL, Gamez J, Gamez J, Vila MR, Kunimatsu T, et al. A novel autosomal dominant limb-girdle muscular dystrophy (LGMD 1F) maps to 7q32.1-32.2. Neurology 2003;61:404–406. 3. Torella A, Fanin M, Mutarelli M, Peterle E, Del Vecchio Blanco F, Rispoli R, et al. Next-generation sequencing identifies transportin 3 as the causative gene for LGMD1F. PLoS One 2013;8:1–7. 4. Melia MJ, Kubota A, Ortolano S, Vılchez JJ, Gamez J, Tanji K, et al. Limb-girdle muscular dystrophy 1F is caused by a microdeletion in the transportin 3 gene. Brain 2013;136:1508–1517. 5. Peterle E, Fanin M, Semplicini C, Vilchez Padilla JJ, Nigro V, Angelini C, et al. Clinical phenotype, muscle MRI and muscle pathology of LGMD1F. J Neurol 2013;260:2033–2041. Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mus.24539 Vincenzo Nigro, MD, PhD Corrado Angelini, MD1,2 2 Letter to the Editor --------------------------------------------------------- MUSCLE & NERVE Month 2015 25 May 2014 Acta Myologica • 2014; XXXIII: p. 1-12 InvIted revIew Genetic basis of limb-girdle muscular dystrophies: the 2014 update Vincenzo Nigro and Marco Savarese Dipartimento di Biochimica, Biofisica e Patologia Generale, Seconda Università degli Studi di Napoli and Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy and the respiratory muscles. The clinical course and the expressivity may be variable, ranging from severe forms with rapid onset and progression to very mild forms allowing affected people to have fairly normal life spans and activity levels (1). The term LGMD is becoming descriptive and also comprises clinical pictures of different diseases. The original definition was given as muscular dystrophies milder that DMD and inherited as autosomal traits (2). However, the most severe forms with childhood onset also result in dramatic physical weakness and a shortened life-span. The advent of next generation sequencing approaches has accelerated the pace of discovery of new LGMD genes. Ten years ago the list included 16 loci (3), while today the LGMD loci so far identified are thirty-one, eight autosomal dominant and 23 autosomal recessive. Limb-girdle muscular dystrophies (LGMD) are a highly heterogeneous group of muscle disorders, which first affect the voluntary muscles of the hip and shoulder areas. The definition is highly descriptive and less ambiguous by exclusion: non-Xlinked, non-FSH, non-myotonic, non-distal, nonsyndromic, and non-congenital. At present, the genetic classification is becoming too complex, since the acronym LGMD has also been used for a number of other myopathic disorders with overlapping phenotypes. Today, the list of genes to be screened is too large for the gene-by-gene approach and it is well suited for targeted next generation sequencing (NGS) panels that should include any gene that has been so far associated with a clinical picture of LGMD. The present review has the aim of recapitulating the genetic basis of LGMD ordering and of proposing a nomenclature for the orphan forms. This is useful given the pace of new discoveries. Thity-one loci have been identified so far, eight autosomal dominant and 23 autosomal recessive. The dominant forms (LGMD1) are: LGMD1A (myotilin), LGMD1B (lamin A/C), LGMD1C (caveolin 3), LGMD1D (DNAJB6), LGMD1E (desmin), LGMD1F (transportin 3), LGMD1G (HNRPDL), LGMD1H (chr. 3). The autosomal recessive forms (LGMD2) are: LGMD2A (calpain 3), LGMD2B (dysferlin), LGMD2C (γ sarcoglycan), LGMD2D (α sarcoglycan), LGMD2E (β sarcoglycan), LGMD2F (δ sarcoglycan), LGMD2G (telethonin), LGMD2H (TRIM32), LGMD2I (FKRP), LGMD2J (titin), LGMD2K (POMT1), LGMD2L (anoctamin 5), LGMD2M (fukutin), LGMD2N (POMT2), LGMD2O (POMTnG1), LGMD2P (dystroglycan), LGMD2Q (plectin), LGMD2R (desmin), LGMD2S (TRAPPC11), LGMD2T (GMPPB), LGMD2U (ISPD), LGMD2V (Glucosidase, alpha ), LGMD2W (PINCH2). Autosomal dominant LGMD The LGMD1, i.e. the autosomal dominant forms, have usually an adult-onset and are milder, because affected parents are usually in quite good health at reproductive age. They are relatively rare representing less than 10% of all LGMD. Sometimes, they correspond to particular cases of mutations in genes involved in other disorders, such as myotilin, lamin A/C or caveolin 3 (Table 1). Key words: Limb-girdle muscular dystrophies, LGMD, NGS LGMD1A - LGMD1A may be caused by mutations in the myotilin (MYOT) gene at chr. 5q31.2. The cDNA is of 2.2 kb and contains 10 exons. Myotilin is a Z-diskassociated protein. LGMD1A may be considered as an occasional form of LGMD (4). The first clinical report was in 1994 (5). The gene was identified in 2000 (6), but myotilin mutations have been rather associated with myofibrillar myopathy. LGMD1A is characterized by late Introduction The term limb-girdle muscular dystrophy refers to a long list of Mendelian disorders characterized by a progressive deterioration of proximal limb muscles. Very often, other muscles are affected, together with the heart Address for correspondence: Vincenzo Nigro, via Luigi De Crecchio 7, 80138 Napoli, Italy; Telethon Institute of Genetics and Medicine (TIGEM), via Pietro Castellino 111, 80131 Napoli, Italy. - E-mail: [email protected] 1 26 May 2014 Vincenzo Nigro and Marco Savarese Table 1. Autosomal dominant limb girdle muscular dystrophy. Gene Clinical phenotype Disease Locus Name Exons LGMD1A 5q31.2 TTID 10 Protein (protein function) Typical onset myotilin (structural; Z disc) Adulthood Progression Cardiomiopathy sCK Slow Not observed 3-4X Allelic disorders (OMIM, #) Myopathy, myofibrillar, 3 (609200) Myopathy, spheroid body (182920) Cardiomyopathy, dilated, 1A(115200) Charcot-Marie-Tooth disease, type 2B1(605588) Emery-Dreifuss muscular dystrophy 2, AD(181350) Emery-Dreifuss muscular dystrophy 3, AR(181350) LGMD1B 1q22 LMNA 12 lamin A/C (structural; fibrous nuclear lamina ) Heart-hand syndrome, Slovenian type(610140) Variable (4-38y) Slow Frequent 1-6X Hutchinson-Gilford progeria(176670) Lipodystrophy, familial partial, 2(151660) Malouf syndrome(212112) Mandibuloacral dysplasia(248370) Muscular dystrophy, congenital(613205) Restrictive dermopathy, lethal(275210) Cardiomyopathy, familial hypertrophic(192600) LGMD1C 3p25.3 CAV3 2 caveolin 3 (scaffolding protein within caveolar membranes) Creatine phosphokinase, elevated serum(123320) Childhood Slow/ moderate Frequent 10X Long QT syndrome 9(611818) Myopathy, distal, Tateyama type(614321) Rippling muscle disease(606072) LGMD1D 7q36 DNAJB6 10 DnaJ/Hsp40 homolog, subfamily B, member 6 (chaperone) Variable (25-50y) Slow Not observed 1-10X Muscular dystrophy, limbgirdle, type 2R(615325) LGMD1E 2q35 DES 9 desmin (structural; intermediate filament) Cardiomyopathy, dilated, 1I(604765) Adulthood Slow Frequent 5-10X Myopathy, myofibrillar, 1(601419) Scapuloperoneal syndrome, neurogenic, Kaeser type(181400) LGMD1F 7q32 TNPO3 23 transportin 3 (nuclear importin) Variable (1-58y) Slow/ moderate Not observed 1-3X - Variable (13-53y) Slow Not observed 1-9X - Variable (10-50y) Slow Not observed 1-10X - LGMD1G 4q21 HNRPDL 9 Heterogeneous nuclear ribonucleoprotein D-like protein (ribonucleoprotein, RNA-processing pathways) LGMD1H 3p23-p25 - - - 2 27 May 2014 Genetic basis of limb-girdle muscular dystrophies: the 2014 update onset proximal weakness with a subsequent distal weakness. Some patients show nasal and dysarthric speech. Serum CK is normal or mildly elevated. Muscle pathology shows rimmed vacuoles with or without inclusions. Electron microscopy shows prominent Z-line streaming. Cardiac and respiratory involvement occasionally occurs. form. LGMD1D is caused by heterozygous missense mutations in the DNAJB6 gene at chr. 7q36.3 (10). The reference cDNA sequence is 2.5kb-long, contains 10 exons and encodes DnaJ homolog, subfamily B, member 6. DNAJ family members are characterized by a highly conserved amino acid stretch (2) called the ‘J-domain’. They exemplify a molecular chaperone functioning in a wide range of cellular events, such as protein folding and oligomeric protein complex assembly (11). Missense heterozygous mutations of DNAJB6 (p.Phe89Ile, p.Phe93Leu and p.Pro96Arg) are all located in the Gly/ Phe-rich domain of DNAJB6 leading to insufficient clearance of misfolded proteins. Functional testing in vivo have shown that the mutations have a dominant toxic effect mediated specifically by the cytoplasmic isoform of DNAJB6. In vitro studies have demonstrated that the mutations increase the half-life of DNAJB6, extending this effect to the wild-type protein, and reduce its protective anti-aggregation effect. DNAJB6 is located in the Z line and interacts with BAG3. Mutations in BAG3 are known to cause myofibrillar myopathy (12). A characteristic pathological finding of LGMD1D is the presence of autophagic vacuoles and protein aggregation. These protein aggregations contain DNAJB6 together with its known ligands MLF1 and HSAP1, and also desmin, αB-crystallin, myotilin, and filamin C, which are known to aggregate in myofibrillar myopathy. These results suggest that the phenotype of LGMD1D also overlaps with that of myofibrillar myopathy. LGMD1D patients show mildly elevated serum CK levels. The lower limbs are more affected, particularly the soleus, adductor magnus, semimembranosus and biceps femoris. In contrast, the rectus femoralis, gracilis and sartorius and the anterolateral lower leg muscles are mostly spared. DNAJB6 gene mutations may also be associated with distal-predominant myopathy. Symptoms in the upper limbs appear later. Some patients develop calf hypertrophy. Onset ranges from 25 to 50 years, with some patients maintaining ambulation throughout life. No cardiac or respiratory involvement has been reported so far. The pattern of differential involvement could be identified at different stages of the disease process. LGMD1B - LGMD1B is also an occasional LGMD form caused by lamin A/C (LMNA) gene mutations at chr. 1q22 (7). The reference cDNA is of 3 kb and contains 12 exons. The LMNA gene gives rise to at least three splicing isoforms (lamin A, C, lamin AΔ10). The two main isoforms, lamin A and C, are constitutive components of the fibrous nuclear lamina and have different roles, ranging from mechanical nuclear membrane maintenance to gene regulation. The ‘laminopathies’ comprise different well-characterized phenotypes, some of which are confined to the skeletal muscles or skin, while others are multi-systemic, such as lipodystrophy, Charcot-Marie Tooth disease, progeroid syndromes, dilated cardiomyopathy and Emery-Dreifuss muscular dystrophy (EDMD). The LGMD1B is characterized by a symmetric proximal weakness starting from the legs, associated with atrioventricular conduction disturbances and dysrhythmias. CK is normal to moderately elevated. Most patients develop proximal leg weakness, followed by cardiac arrhythmias and dilated cardiomyopathy, with sudden death 20-30 years later. However, there is a continuity between LGMD1B and EDMD (8). Usually the more severe forms of EDMD with a childhood onset have missense mutations, whereas the milder LGMD1B is associated with heterozygous truncating mutations: this may arise through a loss of LMNA function secondary to haploinsufficiency, whereas dominant-negative or toxic gain-of-function mechanisms may underlie the EDMD phenotypes. LGMD1C - LGMD1C is caused by mutations in the caveolin 3 gene (CAV3) at chr. 3p25.3. The CAV3 gene encodes a 1.4kb mRNA composed of only two exons. Caveolin-3 is a muscle-specific membrane protein and the principal component of caveolae membrane in muscle cells in vivo: at present this is the only gene in which mutations cause caveolinopathies (9). LGMD1C is characterized by an onset usually in the first decade, a mild-to-moderate proximal muscle weakness, calf hypertrophy, positive Gower sign, and variable muscle cramps after exercise. LGMD1E - For the limb girdle muscular dystrophy originally linked to chr. 6q23 (13) we will use the name LGMD1E, even if it should be considered, more correctly, as a form of autosomal dominant desminopathy or myofibrillar myopathy. This form is also known as dilated cardiomyopathy type 1F (CMD1F). One family previously categorized as having LGMD and dilated cardiomyopathy was reported, indeed, to have the splice site mutation IVS3+3A>G in the desmin (DES) gene at 2q35 (14). LGMD1D - Autosomal dominant LGMD mapped to 7q36 has been classified as LGMD1E in OMIM, but as LGMD1D in the Human Gene Nomenclature Committee Database. In the literature there is another LGMD1D/E erroneously mapped to 6q, but we will use the acronym LGMD1D for the 7q-disease and LGMD1E for the 6q- 3 28 May 2014 Vincenzo Nigro and Marco Savarese Autosomal recessive LGMD For desmin see also LGMD2R. As in the desminopathies, LGMD1E family members show dilated cardiomyopathy and conduction defects together with progressive proximal muscle weakness starting in the second or third decade. Some family members had a history of sudden death. Serum creatine kinase is mildly elevated (150-350U/l). Muscle pathology may show dystrophic changes, but later the presence of abundant perinuclear or subsarcolemmal granulofilamentous inclusions have been also observed. The study of these inclusions by laser capture microdissection followed by mass spectrometry analysis, led to the identification of the disease-causing mutations in desmin (14). The autosomal recessive forms (LGMD2) are much more common, having a cumulative prevalence of 1:15,000 (2) with some differences among countries, depending on the carrier distribution and the degree of consanguinity. There are recessive genes in which the loss-of-function mutations on both alleles tipically result in a LGMD phenotype (ordinary LGMD genes): they correspond to the first 8 forms of LGMD2 (LGMD2A-2H) plus LGMD2L. On the contrary, other genes (occasional LGMD genes) show a phenotypic divergence with some mutations associated with LGMD and other ones determining a more complex disorder. Specific variations in occasional LGMD genes cause the other forms (LGMD2I-2U). The best examples come from dystroglycanopathies in which the LGMD presentation is associated with milder alleles of genes mutated in congenital forms with brain involvement (Table 2). LGMD1F - LGMD1F was originally mapped to a 3.68-Mb interval on chromosome 7q32.1-7q32.2 in a very large Italo-Spanish family. We presented the identification of TNPO3 by whole exome sequencing of four affected family members and the complete refining of the region at the WMS 2012. Data were then published (15): a frame-shift mutation in the transportin 3 (TNPO3) gene is shared by all affected family members with 94% penetrance. The TNPO3 gene is composed of 23 exons and encodes a 923-amino acid protein, also expressed in skeletal muscle. The frame-shifted TNPO3 protein is larger than the wt, since it lacks the predicted stop codon and is found around the nucleus, but not inside. Patients with an onset in the early teens, show a more severe phenotype with a rapid disease course, while adult onset patients present a slower course. They have a prominent atrophy of lower limb muscles, involving especially the vastus lateralis and the ileopsoas muscle (16). Interestingly, some patients present with dysphagia, arachnodactyly and respiratory insufficiency. CK range is 1-3x. No cardiac involvement has been reported. LGMD2A - LGMD2A is caused by Calpain 3 (CAPN3) gene mutations and represents the most frequent LGMD worldwide (20, 21). The CAPN3 gene spans 53kb of genomic sequence at chromosome 15q15.2 and the transcript is composed of 24 exons encoding a 94kDa muscle-specific protein. There is a number of heterozygotes (1:100), carrying many different CAPN3 pathogenic changes. Calpains are intracellular nonlysosomal cysteine proteases modulated by calcium ions. A typical calpain is a heterodimer composed of two distinct subunits, one large (> 80 kDa) and the other small (30 kDa). While only one gene encoding the small subunit has been demonstrated, there are many genes for the large one. CAPN3 is similar to ubiquitous Calpain 1 and 2 (m-calpain and micro-calpain), but contains specific insertion sequences (NS, IS1 and IS2). Calpains cleave target proteins to modify their properties, rather than “break down” the substrates. The phenotypic spectrum of calpainopathies is very broad, but they are true LGMD. For the clinical course, see also (1). LGMD1G - LGMD1G has been mapped to chr. 4q21. Very recently, the defect in the RNA processing protein HNRPDL has been identified (17) in two different families by whole exome sequencing. The HNRPDL gene contains 8 exons and is ubiquiously expressed. The gene product is a heterogeneous ribonucleoprotein family member, which participates in mRNA biogenesis and metabolism. The reduced hnrpdl in zebrafish prodeces a myopathic phenotype. Patients show late-onset LGMD associated with progressive fingers and toes flexion limitation (18). LGMD2B - It is caused by missense or null alleles of the dysferlin (DYSF) gene (22). The DYSF gene spans 233kb of genomic sequence at chr. 2p13.2 and the major transcript is composed of 6,911 nt containing 57 exons in the HGVS recommended cDNA Reference Sequence. Dysferlin is an ubiquitous 230-KDa transmembrane protein involved in calcium-mediated sarcolemma resealing. LGMD2B is the second most frequent LGMD2 form (1525%) in numerous countries, but not everywhere (23). Muscle inflammation is recognized in dysferlinopathy and dysferlin is expressed in the immune cells. LGMD1H - By studying a large pedigree from Southern Italy, a novel LGMD locus has been mapped on chromosome 3p23-p25.1 (19). Most of patients present with a slowly progressive proximal muscle weakness, in both upper and lower limbs, with onset during the fourthfifth decade of life. 4 29 May 2014 Genetic basis of limb-girdle muscular dystrophies: the 2014 update Table 2. Autosomal recessive limb girdle muscular dystrophy. Gene Clinical phenotype Disease Locus Name Exons Protein product LGMD2A 15q15 CAPN3 24 Calpain 3 LGMD Typical onset Progression Cardiomiopathy phenotype Moderate/ rapid sCK ordinary Adolescence Slow Possible 5-40X Rapid Often severe 10–70X Rarely observed Allelic disorders (OMIM, #) 3–20X Miyoshi muscular dystrophy 1 (254130) LGMD2B 2p13.2 DYSF 56 Dysferlin ordinary Young adulthood LGMD2C 13q12 SGCG 8 γ-Sarcoglycan ordinary Early childhood LGMD2D 17q21.33 SGCA 10 α-Sarcoglycan ordinary Early childhood Rapid Often severe 10–70X LGMD2E 4q12 SGCB 6 β-Sarcoglycan ordinary Early childhood Rapid Often severe 10–70X LGMD2F 5q33 SGCD 9 δ-Sarcoglycan ordinary Early childhood Rapid Rarely observed 10–70X Cardiomyopathy, dilated, 1L (606685) LGMD2G 17q12 TCAP 2 Telethonin ordinary Adolescence Slow Possible 10X Cardiomyopathy, dilated, 1N (607487) LGMD2H 9q33.1 TRIM32 2 Tripartite motif containing 32 ordinary Adulthood Slow Not observed 10X Bardet-Biedl syndrome 11 (209900) LGMD2I 19q13.3 FKRP 4 Fukutin related protein ordinary Late childhood Moderate Possible 10-20X Myopathy, distal, with anterior tibial onset (606768) Cardiomyopathy, dilated, 1G (604145) Cardiomyopathy, familial hypertrophic, 9 (613765) LGMD2J 2q24.3 TTN 312 or more Titin occasional Young adulthood Severe Not observed 10-40X Myopathy, early-onset, with fatal cardiomyopathy (611705) Myopathy, proximal, with early respiratory muscle involvement (603689) Tibial muscular dystrophy, tardive (600334) Muscular dystrophydystroglycanopathy (congenital with brain and eye anomalies), type A, 1 (236670) LGMD2K 9q34.1 POMT1 20 Protein-O-mannosyl transferase 1 occasional Childhood Slow Not observed Muscular dystrophy10-40X dystroglycanopathy (congenital with mental retardation), type B, 1 (613155) Muscular dystrophydystroglycanopathy (limbgirdle), type C, 1 (609308) LGMD2L 11p13-p12 ANO5 22 Anoctamin 5 ordinary Variable (young to late adulthood) Slow Not observed 1-15X Gnathodiaphyseal dysplasia (166260) Miyoshi muscular dystrophy 3 (613319) Cardiomyopathy, dilated, 1X (611615) LGMD2M 9q31 FKTN 11 Fukutin occasional Early childhood Moderate Possible Muscular dystrophydystroglycanopathy (congenital with brain and eye anomalies), type A, 4 10-70X (253800) Muscular dystrophydystroglycanopathy (congenital without mental retardation), type B, 4 (613152) (continues) 5 30 May 2014 Vincenzo Nigro and Marco Savarese Table 2. (follows). Gene Disease LGMD2N LGMD2O Locus 14q24 1p34.1 Name POMT2 POMGnT1 Clinical phenotype Exons 21 22 Protein product Protein-O-mannosyl transferase 2 Protein O-linked mannose beta1,2-Nacetylglucosaminyl transferase LGMD Typical onset Progression Cardiomiopathy phenotype occasional Early childhood Slow Rarely observed sCK 5-15X Allelic disorders (OMIM, #) Muscular dystrophydystroglycanopathy (congenital with brain and eye anomalies), type A, 2 (613150) Muscular dystrophydystroglycanopathy (congenital with mental retardation), type B, 2 (613156) Muscular dystrophydystroglycanopathy (congenital with brain and eye anomalies), type A, 3 (253280) occasional Late childhood Moderate Not observed 2-10X Muscular dystrophydystroglycanopathy (congenital with mental retardation), type B, 3 (613151) Muscular dystrophydystroglycanopathy (limbgirdle), type C, 3 (613157) LGMD2P 3p21 DAG1 3 Dystroglycan singular Early childhood Moderate Not observed 20X Epidermolysis bullosa simplex with pyloric atresia (612138) LGMD2Q 8q24 PLEC1 32 Plectin singular Early childhood Slow Not observed Epidermolysis bullosa 10-50X simplex, Ogna type (131950) Muscular dystrophy with epidermolysis bullosa simplex (226670) Muscular dystrophy, limbgirdle, type 2R(615325) LGMD2R 2q35 DES 9 Desmin (structural; intermediate filament) occasional Young adulthood A-V conduction block Cardiomyopathy, dilated, 1I(604765) 1X Myopathy, myofibrillar, 1(601419) Scapuloperoneal syndrome, neurogenic, Kaeser type(181400) LGMD2S 4q35 TRAPPC11 30 Transport protein particle complex 11 occasional Young adulthood Slow Not observed 9-16X Muscular dystrophydystroglycanopathy (congenital with brain and eye anomalies), type A, 14 (615350) LGMD2T 3p21 GMPPB 8 GDP-mannose pyrophosphorylase B occasional Early childhoodYoung adulthood LGMD2U 7p21 ISPD 10 Isoprenoid synthase domain containing occasional Early / Late Rapid/ Moderate Possible 6-50X Muscular dystrophydystroglycanopathy (congenital with brain and eye anomalies), type A, 7 (614643) LGMD2V 17q25.3 GAA 20 Alpha-1,4-glucosidase occasional Variable Variable (Rapid to slow) Possible 1-20X Glycogen storage disease II (232300) LGMD2W 2q14 LIMS2 7 Lim and senescent cell antigen-like domains 2 Childhood - Possible - ? Possible Muscular dystrophydystroglycanopathy (congenital with mental retardation), type B, 14 (615351) 6 31 May 2014 Genetic basis of limb-girdle muscular dystrophies: the 2014 update named adhalin and contains a “dystroglycan-type” cadherin-like domain that is present in metazoan dystroglycans (35). LGMD2E - The beta-sarcoglycan gene spans 15kb of genomic sequence at chromosome 4q11 and the major transcript is composed of 6 exons. The protein contains of 318 amino acids and weighs 43kDa. LGMD2F - Delta-sarcoglycan is by far the largest LGMD gene, spanning 433kb of genomic sequence at chromosome 5q33.3 and the major transcript is composed of 9 exons. Intron 2 alone spans 164kb, one the largest of the human genome. Delta and gamma sarcoglycan are homologous and of identical size (35kDa). LGMD2G - Mutations in titin cap (Tcap)/Telethonin cause LGMD2G, one of the rarest forms of LGMD (36). Tcap provides links to the N-terminus of titin and other Zdisc proteins. Patients show adolescence-onset weakness initially affecting the proximal pelvic muscles and then the distal legs with calf hypertrophy. A homozygous nonsense mutation in the TCAP gene has been described in patient a congenital muscular dystrophy. The TCAP gene has also been associated with cardiomyopathy (37), while common variants may play a role in genetic susceptibility to dilated cardiomyopathy. Immunofluorescence and Western blot assays may show a Telethonin deficiency. Full sequencing testing may be cost-effective in all cases, because the gene is composed only of two small exons. The telethonin gene (TCAP) spans 1.2kb of genomic sequence at chromosome 17q12 and the transcript is composed of 2 exons. The protein product is a 19kDa protein found in striated and cardiac muscle. It binds to the titin Z1-Z2 domains and is a substrate of titin kinase, interactions thought to be critical for sarcomere assembly. Only two different mutations have been described in the TCAP gene in Brazilian patients (36). A mutation (R87Q) was found in a patient with dilated cardiomyopathy (37). Moreover, a human muscle LIM protein (MLP) mutation (W4R) associated with dilated cardiomyopathy (DCM) results in a marked defect in Telethonin interaction/localization (38). LGMD2H - The Tripartite-motif-containing gene 32 (TRIM32) gene spans 14kb of genomic sequence at chromosome 9q33.1 and the transcript is composed of 2 exons, with the first noncoding and the second encoding a 673 aa protein of 72kDa. TRIM32 is a ubiquitous E3 ubiquitin ligase that belongs to a protein family comprising at least 70 human members sharing the tripartite motif (TRIM). The TRIM motif includes three zinc-binding domains, a RING, a B-box type 1 and a B-box type 2, and a coiled-coil region. The protein localizes to cytoplasmic bodies. Although the function of TRIM32 is unknown, analysis of the domain structure of this protein suggests that it may be an E3-ubiquitin ligase (39). The “dysferlinopathies” include limb-girdle muscular dystrophy type 2B (LGMD2B) and the allelic forms Miyoshi myopathy (MM), which is an adult-onset distal form, and distal myopathy with anterior tibialis onset (DMAT), but varied phenotypes are observed. LGMD2B affects earlier the proximal muscles of the arms whereas MM affects the posterior muscles of the leg. DYSF gene mutations are associated with heterogeneous clinical pictures ranging from severe functional disability to mild late-onset forms (24). About 25% of cases are clinically misdiagnosed as having polymyositis (25). This classification into separate phenotypes does not reveal true disease differences (26) and the allelic forms are not due to different mutations. Additional factors (e.g., additional mutations in neuromuscular disease genes or sport activities that include maximal eccentric contractions) may worsen the disease expression of causative mutations in dysferlinopathies (27). WB analysis is very useful and specific (28) when < 20% level of Dysferlin has been identified, although Dysferlin can also be increased or secondarily reduced. NGS-based testing is preferred due to the huge number of exons to be screened and the lack of mutational hot-spots. mRNA analysis also works from blood, albeit with some splice differences (29). LGMD2C-D-E-F Loss-of-function mutations in any of the genes encoding the four members of the skeletal muscle sarcoglycan complex, alpha, beta, gamma and delta-sarcoglycan cause LGMD2D, 2E, 2C and 2F, respectively (30-33). Sarcoglycans are components of the dystrophin-complex. They are all N-glycosylated transmembrane proteins with a short intra-cellular domain, a single transmembrane region and a large extra-cellular domain containing a cluster of conserved cysteines. Sarcoglycanopathies have a childhood onset, similar to intermediate form of Duchenne/Becker dystrophies, and involve both cardiac and respiratory functions. We consider the possibility to classify these forms apart from the other LGMD. LGMD2C - The gamma-sarcoglycan gene spans 144kb of genomic sequence at chromosome 13q12.12 and the transcript is composed of 8 exons. LGMD2C is common in the Maghreb and India (34) for the high allele frequency of 525delT and in gypsies for the C283Y allele. LGMD2C patients may show the absence of y-sarcoglycan together with traces of the other non-mutated sarcoglycans. LGMD2D - The alpha-sarcoglycan gene spans 10kb of genomic sequence at chromosome 17q21.33 and the major transcript is composed of 10 exons. The protein product of 387 amino acids and 50kDa was originally 7 32 May 2014 Vincenzo Nigro and Marco Savarese LGMD2J - TTN is one of the most complex human genes. The titin gene spans 294,442 bp of genomic sequence at chromosome 2q31 and the major transcript is composed of 363 exons. It encodes the largest protein of the human genome composed of 38,138 amino acids with a physical length of 2 microns. An 11-bp indel mutation in the last titin exon causes tibial muscular dystrophy and Gerull et al. (49) showed that a 2-bp insertion in exon 326 of the TTN gene causes autosomal dominant dilated cardiomyopathy (CMD1G; 604145). A homozygous mutation in the C terminus of titin (FINmaj 11bp deletion/ insertion) causes LGMD2J (50). Titin is the giant sarcomeric protein that forms a continuous filament system in the myofibrils of striated muscle, with single molecules spanning from the sarcomeric Z-disc to the M-band (51). Other “titinopathic” clinical phenotypes are tibial muscular dystrophy (TMD, Udd myopathy) (52) or more severe cardiac and muscular phenotypes (53). CAPN3 binds M-band titin at is7 within the region affected by the LGMD2J mutations and shows a secondary deficiency in the LGMD2J muscle (54). Interactions with titin may protect CAPN3 from autolytic activation and removal of the CAPN3 protease reverses the titin myopathology (55). The French nonsense mutation (Q33396X) located in Mex6, seems to cause a milder phenotype than the typical FINmaj mutation (51). Due to the huge gene size, NGS sequencing is the only possible way to study this gene. However, the high number of variants and polymorphisms may have a confounding effect on the diagnosis. LGMD2K - LGMD2K is caused by hypomorphic missense mutations in the POMT1 gene at 9q34, containing 20 exons and spanning about 20 kb. Mutations allowing a residual enzyme activity are linked to mild forms. Different POMT1 alleles, cause congenital muscular dystrophies due to defects of the dystroglycan glycosylation (MDDGC1) and including severe forms with brain and eye anomalies or mental retardation (56-58). LGMD2L - LGMD2L is caused by mutations in the anoctamin-5 (ANO5) gene at 11p14.3 (59). The ANO5 gene spans 90,192 bp and contains 22 exons; the coding sequence is 2.7kb for 913 amino acids. Alternative gene names are TMEM16E and GDD1. Anoctamins are a family of calcium-activated chloride channels (60). This form of LGMD2 is one of the most frequent in Northern Europe encompassing 10%-20% of cases (61). The penetrance is probably incomplete, since females are less frequently affected than males. The most common mutation in Northern Europe is c.191 dupA in exon 5 (62). Patients are usually ambulant and the onset is in adulthood. They show asymmetric muscle involvement with prevalent quadriceps atrophy and pain following exercise. CK levels are 5-20x. There is no evidence for contractures, LGMD2H is usually a late-onset condition characterized by proximal weakness, atrophy, and moderately raised levels of creatine kinase. Until 2008, the only LGMD2H mutation was Asp487Asn found in Hutterite families (40). Different TRIM32 mutations were then identified in Italian LGMD patients (41) that accounts for about 3% of LGMD2. The D487N mutation of TRIM32 causes the more severe sarcotubular myopathy (STM). Recently, two other LGMD2H patients have been described associated with STM morphotype (42). LGMD2I, LGMD2K, LGMD2M, LGMD2N, LGMD2O, and LGMD2P The name dystroglycanopathy has been given to defects due to mutations in six genes (POMT1, POMT2, POMGnT1, FKTN, FKRP and DAG1) (43). These variations reduce dystroglycan glycosylation and cause a wide range of phenotypes ranging from mild congenital muscular dystrophies to dramatic conditions, including brain and eye anomalies (muscle–eye–brain disease or Walker– Warburg syndrome). LGMD2I - The fukutin-related protein gene spans 12kb of genomic sequence at chromosome 19q13.32 and the transcript is composed of 4 exons, with the first three noncoding. The extracellular part of the dystrophin/ utrophin-associated complex is also involved in congenital muscular dystrophies, as well as in LGMD2I. Fukuyama-type congenital muscular dystrophy (FCMD), is one of the most common autosomal recessive disorders in Japan characterized by a congenital muscular dystrophy associated with brain malformation (micropolygria) due to a defect in the migration of neurons caused by mutation in the fukutin gene at 9q31 (44). Mutations in the fukutin-related protein gene (FKRP) at 19q13 cause a form of congenital muscular dystrophy with secondary laminin alpha2 deficiency and abnormal glycosylation of alpha-dystroglycan (45). The same gene is also involved in LGMD2I (15). All of these diseases are associated with changes in alpha-dystroglycan expression due to a glycosylation defect of alpha-dystroglycan. Dystroglycan is normally expressed and recognized by polyclonal antibodies, but it is abnormally glycosylated and not recognized by monoclonal antibodies directed against certain epitopes. FKRP is resident in the Golgi apparatus. The P448L mutation, that results in CMD1C, causes a complete mislocalization of the protein and the alpha-dystroglycan is not processed, while LGMD2I mutations affect the putative active site of the protein or cause inefficient Golgi localization (46). LGMD2I mutations appear to be a relatively common cause of LGMD, accounting for at least 10% of all LGMD with either severe or mild phenotypes (47, 48). 8 33 May 2014 Genetic basis of limb-girdle muscular dystrophies: the 2014 update specific transcript Plectin 1f), while there are many other alternative first exons that are spliced to a common exon 2. These patients produce normal skin plectin and do not show skin pathology. LGMD2Q patients show early-onset non-progressive or slowly progressive LGMD. LGMD2R - Desmin is the muscle-specific member of the intermediate filament (IF) protein family (76). The desmin (DES) gene at 2q35 contains 9 exons and spans about 8.4 kb. It encodes a 468-amino acid protein. Autosomal dominant mutations in the DES gene are associated with myofibrillar myopathy (14). The overlap with the DES gene has also been claimed for LGMD1E (77). A homozygous splice site mutation has been identified in two Turkish sibs, born of consanguineous parents, in intron 7 of the DES gene (c.1289-2A>G), resulting in the addition of 16 amino acids from residue 428. Since then, other mutations have been identified. The patients have onset in their teens or twenties of progressive proximal muscle weakness and non-specific atrophy affecting both the upper and lower limbs. The serum Ck is normal. LGMD2R patients usually show A-V conduction blocks but no cardiomyopathy. LGMD2S - This is caused by mutation in the transport protein particle complex 11 (TRAPPC11) gene that spans 54,328 bp at chr. 4q35, the mRNA is 4.5kb and contains 30 exons. Recently, mutations in TRAPPC11 have been identified in a consanguineous Syrian family with an uncharacterized form of LGMD and in five Hutterite individuals presenting with myopathy, ID, hyperkinetic movements and ataxia (78). TRAPPC11 is a transport protein particle component involved in anterograde membrane transport from the endoplasmic reticulum (ER) to the ER-to-Golgi intermediate compartment (ERGIC) in mammals (79). Mutations identified so far (c.2938G>A/ p.Gly980Arg and c.1287+5G>A) cause modifications in TRAPP complex composition, in Golgi morphology and in cell trafficking. The LGMD2S pathogenic mechanism is similar to that causing Danon disease, an X-linked myopathy due to LAMP2 mutations and affecting the secretory pathway (80). The LGMD2S phenotype ranges from a slowly progressive LGMD with childhood onset and high CK to a syndrome characterized by myopathy but also neurological involvement (ID and ataxia). LGMD2T - LGMD2T is caused by milder mutations in the GDP-mannose pyrophosphorylase B (GMPPB) gene (81). The GMPPB gene is a small gene of 2,453bp at chr. 3p21. The mRNA is 1.7kb and contains 8 exons. Mutations in the GMPPB gene have been associated with congenital muscular dystrophies with hypoglycosylation of α-dystroglycan and also with LGMD only in three un- cardiomyopathy or respiratory involvement. LGMD2L is allelic with the AD gnathodiaphyseal dysphasia (63) and with AR distal myopathy (MMD3) (64). LGMD2M - This is associated with mutations in the fukutin gene (FKTN) at chr. 9q31.2 (65). The FKTN gene spans 82,989 bp and contains 10 coding exons, the main transcript is 7.4kb encoding a protein of 413 amino acids. Also in this case LGMD2M is a milder form caused by at least one hypomorphic missense mutation in a gene that, with both non-functional alleles, is associated with more severe phenotypes (66): WWS, MEB or congenital muscular dystrophies (67). In LGMD2M the CNS is not affected and the intelligence is normal. Patients are hypotonic, may be ambulant and the onset is in early childhood. They show symmetric and diffuse muscle involvement that deteriorates with acute febrile illness. Improvement is seen with steroids. CK levels are 10-50x. There is also evidence for spinal rigidity, contractures and cardiomyopathy and respiratory involvement. LGMD2N - Mutations in the POMT2 gene, containing 21 exons, at chr. 14q24 cause LGMD2N (68). POMT2 is a second O-mannosyltransferase overlapping with POMT1 expression. POMT2 mutations usually have a dramatic effect: they cause Walker-Warburg syndrome or muscle-eye-brain-like (69), but rarely are associated with LGMD (70). This may occur when the α-dystroglycan glycosylation is only slightly reduced. In these cases the mutations are usually missense and the phenotype is characterized by LGMD without brain involvement, very high serum CK. LGMD2O - It is associated with milder mutations in the POMGnT1 gene at chr. 1p32 (71). Usually mutations in the POMGnT1 gene are associated with more severe phenotypes than LGMD, such as Walker-Warburg syndrome or MEB. A homozygous hypomorphic allele of the POMGnT1 gene was found as a 9-bp promotor duplication (72). LGMD2P - LGMD2P is caused by specific changes of the dystroglycan (DAG1) gene itself. Recently, Campbell has reported a missense mutation in the dystroglycan gene in an LGMD patient with cognitive impairment (73). This substitution interferes with LARGE-dependent maturation of phosphorylated O-mannosyl glycans on α-dystroglycan affecting its binding to laminin. As a rule the dystroglycanopathies are due to mutations in genes involved in the glycosylation pathway of dystroglycan, but the dystroglycan gene is normal. LGMD2Q - This form of LGMD is mutation-specific since other mutations in the Plectin (PLEC1) gene at chrom. 8q24.3 cause epidermolysis bullosa simplex (74). LGMD2Q has been identified as a homozygous 9-bp deletion in consanguineous Turkish families (75). The deletion affects an AUG that is only present in a muscle- 9 34 May 2014 Vincenzo Nigro and Marco Savarese related patients so far reported. The patients from Indian and Egyptian descent presented with microcephaly and intellectual delay. All 3 patients had increased serum creatine kinase and dystrophic findings on muscle biopsy. Muscle biopsy showed hypoglycosylation of DAG1. The English LGMD patient was a 6-year-old boy with exercise intolerance and CK = 3,000 UI. Two missense mutations were identified: pAsp27His and p.Val330Ile. LGMD2U - This is the form caused by some particular alleles of the isoprenoid synthase domain containing (ISPD) gene. The ISPD gene spans 333kb at chromosome 7p21 and contains 10 exons. ISPD mutations disrupt dystroglycan mannosylation and cause of WalkerWarburg syndrome (82, 83). Mutations in ISPD as well as TMEM5 genes have been associated with severe cobblestone lissencephaly (84). Null alleles of ISPD produce Walker Warburg or cobblestone lissencephaly with brain vascular anomalies, but at least one milder mutation in one allele has been found in LGMD (68 69). We named this forms as LGMD2U. The association between mutations in the ISPD gene and LGMD was, however, older than that of forms 2P-2T, but to avoid discordant definitions among the LGMD2U should be considered as that caused by some alleles of ISPD. LGMD2U is progressive, with most cases with LGMD losing ambulation in their early teenage years, thus following a DMD-like path. In several patients, there is muscle pseudohypertrophy, including the tongue. Respiratory and cardiac functions also decline, resembling other dystroglycanopathies. LGMD2V - This is a proposal to name as LGMD2V an occasional LGMD form that derives from mild mutations of the acid alpha-glucosidase (GAA) gene (85). The GAA gene maps at chr 17q25.3 and comprises 20 exons with a protein product of 953 aa. Defects in GAA are the cause of glycogen storage disease type 2 (GSD2, MIM: 232300). GSD2 is a metabolic disorder with a broad clinical spectrum. The severe infantile form, or Pompe disease, presents at birth with massive accumulation of glycogen in muscle, heart and liver. Late-onset Pompe disease may present from the second to as late as the seventh decade of life with progressive proximal muscle weakness primarily affecting the lower limbs, as in a limb-girdle muscular dystrophy. Final outcome depends on respiratory muscle failure. LGMD2W - This caused by mutations in the LIM and senescent cell antigen-like-containing domain protein 2 (LIMS2/ PINCH2) gene at chromosome 2q14. The gene comprises 7 coding exons. It encodes a 341-aa member of a small family of focal adhesion proteins. The encoded protein has five LIM domains, each domain forming two zinc fingers, which permit interactions which regulate cell shape and migration. Patients show a childhood onset LGMD with macroglossia and calf enlargement. They al- so developed decreased ejection fraction with global left ventricular dysfunction in their 3rd decade, severe quadriparesis and relative sparing of the face, and characteristically a broad based triangular tongue. This form has been presented in a poster session at the ASHG 2013. The classification of LGMD is becoming too complex. We tried to reorganize the different genes so far described following the traditional nomenclature. However for the autosomal recessive forms there are few letters available. 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Whole-body T1 weighted MRI was performed in order to evaluate the muscle involvement pattern respectively in one mildly and one severely affected patients. 27 patients (15M–12F, 9–66 years) from 22 families were included. Two populations could be identified according to disease severity: a severe form (n = 17) with onset <10 years (median 3 years) and early loss of ambula-tion (13/17pts, median 12 years) and a milder form (n = 10) with later onset (median 13.5 years) and slower progression (two patients ambulant at 50 and 66 years). Fifty-one mutated alleles were identified (14 muta-tions) in 26 patients; two mutations were recurrent, associated with the severe form (c.376_383dup, 13/34 alleles) or the milder form (c.-22_10dup32, 8/20). A hypokinetic or dilated cardiomyopathy was observed in 12 patients (44%, median 28.5 years). Six patients had a restrictive respiratory insufficiency requiring ventilation (22%, median 39 years). MRI examinations showed similar area of fatty replacement more pronounced in the older patient with longer evolution: Latissimus dorsi, spine extensors and abdominal belt in trunk; glutei, great and lon-gus adductors in pelvic girdle; anterior and posterior compartments with sparing of rectus femoris, gracilis, sartorius and short head of the biceps femoris in thighs. This study refines the phenotypic spectrum of LGMD2E, and identifies two mutations predictive of the disease course. The LGMD2E phenotype is associated with a high incidence of cardiomy-opathy and less frequent respiratory insufficiency. http://dx.doi:10.1016/j.nmd.2013.06.459 P.5.8 Why is LGMD2G rare? C.F. Almeida 1, P.C.G. Onofre-Oliveira 2, M. Zatz 2, L. Negrao 3, M. Vainzof 2 1 Human Genome Research Center, Institute of Biosciences, University of Sã o Paulo, Genetics and Evolutionary Biology, Sã o Paulo, Brazil; 2 University of Sã o Paulo, Genetics and Evolutionary Biology, Sã o Paulo, Brazil; 3 Coimbra’s University Hospital, Coimbra, Portugal Mutations in telethonin gene cause a rare and relatively mild form of limb-girdle muscular dystrophy type 2G. Only few families were described presenting this disease, and they are mainly Brazilians. In Brazil, this form represents less than 5% of all LGMD. In other countries, only isolated sporadic cases were described in China, Moldavia, Australia and Portugal. To date, all ten families identified in Brazil present the same c.157C > T (Q53X) homozygous nonsense mutation. In five families no consanguinity was referred. All patients also share the same haplotype for microsatellite markers near the gene, suggesting a common origin of the mutation. Outside Brazil, different mutations were identified in China, Moldavia and Australian. However, the Portuguese patient described presents the same mutation found in Brazilians. As a great proportion of Brazilian people has Portuguese ancestry, and the Brazilian LGMD2G patients show a predominant European genetic background, we consider that the common mutation arose in Europe and spread through Brazilian population. However, in this case, it would still be expected to find a higher frequency of this disease in Portugal. By sequencing TCAP exon 2, we found that all the patients, including the Portuguese one, are homozygous for the allele A of rs1053651 SNP (the ancestral allele is C). This allele has a frequency of 28% in the European population. Thus, we hypothesize that the muta-tion may have occurred in this rarer haplotype, which could explain why this form is also rare in Portugal. Therefore, the c.157C > T mutation has a common origin, that implies the occurrence of founder effect, probably in Portugal and is in linkage disequilibrium with the rs1053651 SNP, which is compatible with its low frequency in Europe. Financial support: FAPESPCEPID, CNPQ-INCT, FINEP, CAPES-COFECUB. http://dx.doi:10.1016/j.nmd.2013.06.460 P.5.9 Clinical and molecular analysis of a large cohort of patients with anoctaminopathy A. Sarkozy 1, D. Hicks 1, J. Hudson 1, S.H. Laval 1, R. Barresi 2, M. Guglieri 1, E. Harris 1, V. Straub 1, K. Bushby 1, H. Lochmuller 1 1 Institute of Genetic Medicine, International Centre for Life, Newcastle upon Tyne, United Kingdom; 2 NSCT Diagnostic & Advisory Service for Rare Neuromuscular Diseases, Muscle Immunoanalysis Unit, Dental Hospital, Newcastle upon Tyne, United Kingdom Recessive mutations in the ANO5 gene cause a spectrum of phenotypes ranging from isolated hyperCKaemia to limb girdle muscular dystrophy (LGMD2L), characterized by adult onset proximal lower limb muscular weakness and raised CK values. The recurrent exon 5 mutation (c.191dupA) has been found in most of the British and German patients so far reported. We performed molecular analysis of the ANO5 gene in a large cohort of undiagnosed patients with clinical suspicion of anoctaminopathy. We identified two pathogenic mutations in 42/205 unrelated patients (21%), while a single change only was found in further 14 patients. Fifteen pathogenic changes were novel. The founder c.191dupA mutation represents 61% of mutated alleles but is confirmed to be less prevalent in non-Northern European populations. Retrospective clinical analysis of patients with 2 mutations corroborates previous finding such as the male predominance and absence of major cardiac or respiratory involvement, as well as very mild late onset cases of both sexes and isolated hyperCKaemia only. Our results also confirm anoctaminopathy as one of the most common adult muscular dystrophies in Northern Europe, with a prevalence of about 20–25% in undiagnosed patients. http://dx.doi:10.1016/j.nmd.2013.06.461 P.5.10 Clinical and ultrastructural changes in transportinopathy C. Angelini 1, E. Peterle 1, M. Fanin 1, G. Cenacchi 2, V. Nigro 3 1 University of Padova, Padova, Italy; 2 University of Bologna, Bologna, Italy; 3 TIGEM, Napoli, Italy Muscle histopathological, ultrastructural and genetic features of a large Italian-Spanish family with autosomal dominant LGMD, previously mapped to 7q32.1–32.2 (LGMD1F) were studied in 3 biopsies. We collected the clinical history in 19 of 60 patients; muscle biopsy histopathology was investigated in one pair of affected patients (mother 1 biopsy, her daughter 2 consecutive biopsies at 9 and 22 years). We observed that the age of onset varied from 2 to 35 years, and occurred either in upper or in the lower girdle; in 14 cases there was hypotrophy both in proximal upper and in lower extremities in calf muscles. The severity was not increased in successive generations. Unreported clinical findings were arachnodactyly, dysphagia and dysarthria. Moreover, we noticed a discrepancy between the clinical severity and muscle biopsy involvement: the daughter has a more severe clinical course, the first biopsy had only type 1 fiber atrophy while increased fiber atrophy was observed in the second biopsy. The mother has a compromised muscle histopathology (more muscle fiber variation, and autophagic changes by acid phosphatase stain). An abnormal sarcomeric assembly is the cause 38 October, 2013 Abstracts / Neuromuscular Disorders 23 (2013) 738–852 767 of progressive atrophy and myofiber loss. Electron microscopy revealed accumulation of myofibrillar bodies in muscle fibers. Accumulation of desmin and myotilin and p62-positive aggregates was observed. A defect in transportin-3 gene has been found to be the cause of this disease, which represents a new mechanism of dominant myopathy. Our morphological and ultrastructural data seems to suggest a phenotype similar to myofibrillar disease; however, autophagosomes were also present. It is possible that SR protein cannot migrate or be transported in- and out-of the nuclear membrane. Autonoma de Barcelona, Research Group on Neuromuscular and Mitochondrial Disorders, Barcelona, Spain; 3 Institute of Biomedical Research of Vigo (IBIV), University Hospital of Vigo (CHUVI), Department of Pathology and Neuropathology, Vigo, Spain; 4 Hospital Universitari i Politecnic La Fe, Department of Neurology, Valencia, Spain; 5 Hospital Universitari Vall dHebron, Institut de Recerca, Universitat Autonoma de Barcelona, Neuromuscular Disorders Clinic,Department of Neurology, Barcelona, Spain; 6 Columbia University Medical Centre, Department of Pathology and Cell Biology, New York, United States; 7 Centro Nacional de Analisis Genomico, Barcelona, Spain http://dx.doi:10.1016/j.nmd.2013.06.462 Limb-girdle muscular dystrophy 1F (LGMD1F) is an autosomal dominant muscular disease affecting a Spanish family. Using whole genome sequencing, we identified a single nucleotide deletion (c.2771del) in transportin-3 gene (TNPO3) in a LGMD1F patient. The mutation disrupts the termination codon of TNPO3 and causes a reading frame shift. Transportin-3 is a nuclear protein, and mediates import of serine–arginine rich proteins into nucleus, which is important for mRNA splicing. This study aimed to investigate the significance of transportin-3 in the pathogenesis of LGMD1F. We performed dideoxy-sequencing of TNPO3 in 24 affected and 23 unaffected family members. Muscle specimens from 4 patients were analyzed by conventional stains and immunohistochemistry. Direct sequence of TNPO3 revealed that all patients carried a heterozygous mutation, and none of the unaffected subjects had the mutation. Hematoxylin-eosin (HE) stained muscle revealed nuclei (10.7 ± 3.0%; mean ± SD) with central pallor in all patients studied. Immunohistochemistry with anti-transportin-3 antibody showed colocalization with nuclei in control subjects. In patients, transportin-3 was also observed within nuclei, but was often unevenly distributed in periphery, a staining pattern similar to that seen by HE. Genetic and histological studies in a Spanish family strongly support the hypothesis that TNPO3 is the causative gene of LGMD1F. Pathological study also indicates that the subcellular distribution of transportin-3 is disrupted and affects the structure of nuclei. P.5.11 LGMD1D mutations in DNAJB6 disrupt disaggregation of TDP-43 R. Bengoechea 1, E.P. Tuck 1, K.C. Stein 2, S.K. Pittman 1, R.H. Baloh 3, H.L. True 2, M.B. Harms 1, C.C. Weihl 1 1 Washington University, Neurology, St Louis, United States; 2 Washington University, Cell Biology and Physiology, St Louis, United States; 3 CedarsSinai Medical Center, Neurology, Los Angeles, United States Heat shock proteins (HSPs) facilitate the folding or degradation of misfolded, damaged and aggregated proteins. Disruptions in HSP function may underlie the molecular basis of many degenerative disorders including some myopathies. The pathogenic mechanism of these chaperonopathies is unclear. We recently identified mutations in DNAJB6, an HSP40 co-chaperone, as the cause of a hereditary IBM also named LGMD1D. One feature of LGMD1D muscle is the accumulation of protein inclusions that contain TDP-43. TDP-43 is an RNA binding protein with a prion-like domain (PrLD) that is mutated in familial amyotrophic lateral sclerosis (ALS). LGMD1D mutations in DNAJB6 reside within the highly conserved G/F domain. Although the role of the G/F domain in DNAJB6 is unclear, studies in S.cerevisiae, have shown that the homologous G/F domain in Sis1 (a DNAJB6 ortholog) is required for the propagation of select yeast prions. Yeast prions contain Q/N rich PrLDs, a feature they share with TDP-43 and other RNA binding proteins. Consistent with this, homologous LGMD1D mutation in the G/F domain of Sis1 abrogate its ability to modulate yeast prion propagation. In mammalian cell culture DNAJB6 associates with TDP-43 in the nucleus upon heat shock suggesting that TDP-43 is indeed a DNAJB6 client protein. DNAJB6 expression reduces the formation and enhances the dissolution of TDP-43 positive nuclear bodies. LGMD1D mutant DNAJB6 expression increases TDP-43 granule formation and slows their dissolution upon heat shock recovery. This effect is more pronounced in cells expressing DNAJB6 that lacks the G/F domain. We hypothesize that LGMD1D mutant DNAJB6 affects localization, aggregation and toxicity of TDP43. Characterization of a transgenic mouse model of LGMD1D recently generated in our laboratory will help to elucidate the role of DNAJB6 and other HSPs in skeletal muscle disease and the complex interplay between RNA binding protein aggregation and disaggregation. http://dx.doi:10.1016/j.nmd.2013.06.463 P.5.12 A mutation in TNPO3 causes LGMD1F and characteristic nuclear pathology A. Kubota 1, M.J. Melia 2, S. Ortolano 3, J.J. Vilchez 4, J. Gamez 5, K. Tanji 6, E. Bonilla 6, L. Palenzuela 2, I. Fernandez-Cadenas 2, A. Pristoupilova 7, E. Garcia-Arumi 2, A.L. Andreu 2, C. Navarro 3, R. Marti 2, M. Hirano 1 1 Columbia University Medical Centre, Department of Neurology, New York, United States; 2 Vall dHebron Institut de Recerca, Universitat http://dx.doi:10.1016/j.nmd.2013.06.464 P.5.13 Remarkable muscle pathology in DNAJB6 mutated LGMD1D S.M. Sandell 1, S. Huovinen 2, J.M. Palmio 1, H. Haapasalo 2, B.A. Udd 1 1 Neuromuscular Research Center, Tampere University Hospital, Neurology, Tampere, Finland; 2 Neuromuscular Research Center, Tampere University Hospital, Pathology, Tampere, Finland Limb girdle muscular dystrophies are a large group of both dominantly and recessively inherited muscle diseases. Dominantly inherited LGMD1 diseases are usually milder and later onset forms than recessive LGMD2. We have followed six Finnish families with LGMD1D and reported clinical and MRI findings in these families. All families represent the same DNAJB6 mutation, causing a F93L change in the ubiquitously expressed co-chaperone DNAJB6. The molecular pathogenesis of LGMD1D is mediated by defective chaperonal function leading to impaired handling of misfolded proteins which normally, without the defect, would be degraded and re-cycled. We have analyzeded 14 muscle biopsies obtained from 13 patients in six families at very different time points after onset of muscle weakness symptoms. All biopsies were from lower limb muscles, either vastus lateralis or gastrocnemius medialis and processed for routine histology, histochemistry as well as extensive immunohistochemistry and semithin sections with subsequent electron microscopy. Uniform findings were myopathic/dystrophic changes in all patients. Restricted and easily overlooked myofibrillar pathology in routine histopathology included protein aggregates reactive for Z-disk proteins such 39 May 7th, 2013 Distrofia dei cingoli, Telethon scopre il gene responsabile della rara patologia 07 Maggio 2013 Ricercatori del Tigem di Napoli chiariscono le basi della distrofia dei cingoli di tipo 1F tramite tecniche di sequenziamento di ultima generazione Napoli - Identificato il difetto genetico alla base di una rara forma di distrofia muscolare dei cingoli, quella di tipo 1F: a descriverlo sulle pagine di Plos One è stato un gruppo di ricercatori dell’Istituto Telethon di genetica e medicina (Tigem) di Napoli, guidati da Vincenzo Nigro, che si sono avvalsi delle più sofisticate tecnologie di sequenziamento del genoma oggi a disposizione. "Come suggerisce anche il nome, questa malattia porta a una progressiva debolezza dei muscoli dei cingoli pelvico e scapolare, compromettendo così la capacità di sollevare pesi e camminare" spiega Nigro. "Riconoscerla e diagnosticarla correttamente, però, non è facile, perché è molto eterogenea sia nella sua manifestazione clinica – età di insorgenza e gravità variano molto da un paziente all’altro – sia dal punto di vista genetico. Ancora oggi, nel 40 per cento dei casi non è possibile identificare lo specifico gene alterato nel paziente: questo non è velleitario, perché una precisa diagnosi molecolare innanzitutto conferma il tipo di patologia, poi dà informazioni su come evolverà nel tempo e permette di effettuare la consulenza genetica agli altri componenti della famiglia". Analizzando così il patrimonio genetico di 64 individui di una famiglia italo-spagnola affetti da una forma di distrofia dei cingoli dalle basi genetiche ancora sconosciute, Nigro e il suo team hanno identificato il responsabile in un gene localizzato sul cromosoma 7, quello di una proteina chiamata Transportina 3. I pazienti con questa mutazione presentano, oltre ai segni tipici della distrofia dei cingoli, debolezza facciale, disfagia, disartria, atrofia e contrattura dei muscoli delle mani, come descritto dai colleghi dell’Università di Padova guidati da Corrado Angelini. L’analisi genetica è stata possibile grazie alle apparecchiature all’avanguardia disponibili presso l’Istituto Telethon di Napoli, quelle per il cosiddetto “next-generation sequencing”. "Grazie a questi approcci di straordinaria potenza oggi possiamo analizzare grandi quantitativi di Dna in tempi relativamente rapidi" continua Nigro. "Basti pensare che lo storico Progetto genoma umano ha richiesto ben 10 anni e 3 miliardi di dollari per arrivare al sequenziamento del patrimonio genetico dell’uomo. Oggi con i nostri macchinari possiamo analizzare in soli dieci giorni la parte codificante del genoma di 48 individui contemporaneamente, per un costo dei reagenti che non supera i 38mila euro. In pratica, il Dna viene spezzettato, selezionato, sequenziato e poi 'ricomposto' al computer per determinare la completa sequenza di lettere". Questo lavoro di analisi è molto delicato e richiede alte competenze di bioinformatica per leggere i dati e trarne delle conclusioni corrette: al Tigem di Napoli ci sono ricercatori specializzati proprio in questo, come Margherita Mutarelli, tra gli autori dello studio. "Il risultato di questo lavoro è importante innanzitutto per le famiglie, cui possiamo finalmente fornire una diagnosi molecolare corretta, ma anche per la ricerca: quello messo in luce è un meccanismo patologico del tutto nuovo, che potrebbe spiegare anche altre malattie simili che colpiscono i muscoli" conclude Nigro. "Il nostro lavoro, grazie anche al supporto di Telethon, continuerà quindi lungo due binari: da un lato chiarire il ruolo della proteina che abbiamo identificato come responsabile della forma 1F di distrofia dei cingoli, dall’altra utilizzare questa stessa tecnologia per andare alla ricerca dei geni responsabili delle forme ancora “orfane” di questa malattia. Ricordiamoci infatti che anche tra le malattie rare ce ne sono alcune più trascurate di altre, per le quali cioè non manca soltanto una cura efficace, ma anche una conoscenza minima di base." 40 May, 2013 Next-Generation Sequencing Identifies Transportin 3 as the Causative Gene for LGMD1F Annalaura Torella1,2., Marina Fanin3., Margherita Mutarelli1, Enrico Peterle3, Francesca Del Vecchio Blanco2, Rossella Rispoli1,4, Marco Savarese1,2, Arcomaria Garofalo2, Giulio Piluso2, Lucia Morandi5, Giulia Ricci6, Gabriele Siciliano6, Corrado Angelini3,7, Vincenzo Nigro1,2* 1 TIGEM (Telethon Institute of Genetics and Medicine), Napoli, Italy, 2 Dipartimento di Biochimica Biofisica e Patologia Generale, Seconda Università degli Studi di Napoli, Napoli, Italy, 3 Dipartimento di Neuroscienze, Università degli Studi di Padova, Padova, Italy, 4 Cancer Research UK, London, United Kingdom, 5 Fondazione IRCCS Istituto Neurologico C. Besta, Milano, Italy, 6 Dipartimento di Medicina clinica e sperimentale, Università degli Studi di Pisa, Pisa, Italy, 7 IRCSS S. Camillo, Venezia, Italy Abstract Limb-girdle muscular dystrophies (LGMD) are genetically and clinically heterogeneous conditions. We investigated a large family with autosomal dominant transmission pattern, previously classified as LGMD1F and mapped to chromosome 7q32. Affected members are characterized by muscle weakness affecting earlier the pelvic girdle and the ileopsoas muscles. We sequenced the whole exome of four family members and identified a shared heterozygous frame-shift variant in the Transportin 3 (TNPO3) gene, encoding a member of the importin-b super-family. The TNPO3 gene is mapped within the LGMD1F critical interval and its 923-amino acid human gene product is also expressed in skeletal muscle. In addition, we identified an isolated case of LGMD with a new missense mutation in the same gene. We localized the mutant TNPO3 around the nucleus, but not inside. The involvement of gene related to the nuclear transport suggests a novel disease mechanism leading to muscular dystrophy. Citation: Torella A, Fanin M, Mutarelli M, Peterle E, Del Vecchio Blanco F, et al. (2013) Next-Generation Sequencing Identifies Transportin 3 as the Causative Gene for LGMD1F. PLoS ONE 8(5): e63536. doi:10.1371/journal.pone.0063536 Editor: Paul McNeil, Medical College of Georgia, United States of America Received February 15, 2013; Accepted March 25, 2013; Published May 7, 2013 Copyright: 2013 Torella et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was mainly supported by grants from Telethon, Italy (TGM11Z06 to V.N. and GTB12001 to C.A.) and Telethon-UILDM (Unione Italiana Lotta alla Distrofia Muscolare) (GUP 10006 and GUP11006 to V.N.). This work was also supported by grants from the Association Française contre les Myopathies (13859 to M.F. and 14999/16216 to C.A.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] . These authors contributed equally to this work. four autosomal dominant LGMD genes are known, encoding Myotilin (LGMD1A), Lamin A/C (LGMD1B), Caveolin-3 (LGMD1C), and DNAJB6 [7,8] (LGMD1D). Some patients with mutations in these four genes fulfill the diagnostic criteria for the LGMDs, but others show a much wider spectrum of different phenotypes. LGMD1F is a very puzzling disease [9]. It is characterized by muscle weakness affecting earlier the pelvic girdle and especially the ileopsoas muscle. Interestingly, some patients presented with a juvenile-onset form. In the original article [9], rimmed vacuoles were reported. Recently, immunofluorescence and ultrastructural studies pointed to the presence of large protein aggregates and autophagosomes [10]. Many alterations of myofibrillar component were also detected [10]. The critical interval was mapped to a 3.68-Mb interval on chromosome 7q32.1–7q32.2 [11]. Given the size of the kindred and the very accurate linkage analysis, the gene identification has been considered within reach. In this region the obvious candidate is the FLNC (Filamin C) gene that is mutated in a form of autosomal dominant myofibrillar myopathy (MFM) with limbgirdle involvement [12], as well as in a second form characterized by the weakness of distal muscles and non-specific myopathic features [13]. However, the early onset of some LGMD1F and the lack of massive protein aggregates of MFM suggest that LGMD1F may be a different disorder: despite a thorough search, no mutation was found in the FLNC gene [11]. In addition, other Introduction Limb girdle muscular dystrophies (LGMDs) are characterized by a progressive weakness that begins from the proximal limb muscles, due to a number of independent genetic defects that are distinct from the X-linked Duchenne and Becker muscular dystrophies [1,2]. In addition to the genetic heterogeneity, the different forms are clinically heterogeneous, with the age at onset of symptoms varying from early childhood to late adulthood [3]. The milder the symptoms are, more difficult is the LGMD diagnosis. Magnetic resonance imaging is helpful to characterise the severity and pattern of muscle involvement [4,5], but recognition of LGMD type might be hard [6]. Muscle biopsy of LGMD patients generally shows a diffuse variation in fiber size, necrosis, regeneration and fibrosis, but the degree of these factors is variable and does not parallel the clinical severity. Based on the histological features alone, there is scarce, if any, possibility of diagnosing a specific LGMD form, but western blot and immunofluorescence can address to the true defect that can be demonstrated by the finding of a mutation in the corresponding gene. The primary distinction is made between the autosomal dominant (LGMD1) and the autosomal recessive forms (LGMD2), with an alphabet letter indicating the order of gene mapping [2]. Eight LGMD1 loci have been identified so far. At present, only PLOS ONE | www.plosone.org 1 May 2013 | Volume 8 | Issue 5 | e63536 41 May, 2013 TNPO3 Is the LGMD1F Gene (local realignment around in-del and base recalibration) and SNV and in-del calling were performed with Genome Analysis Toolkit (GATK) [19]. The called SNV and in-del variants produced with both platforms were annotated using ANNOVAR [20], the relative position in genes using RefSeq [21], amino acid change, presence in dbSNP v137 [22], frequency in NHLBI Exome Variant Server (http://evs.gs.washington.edu/EVS) and 1000 genomes large scale projects (http://www.1000genomes.org) [23], conservation and different prediction algorithms of damaging effect on protein activity [24,25,26,27] and conservation scores [28,29]. The annotated results were then imported into an in-house variation database, also used to make comparisons among samples and filter results. The alignments at candidate positions were visually inspected using the Integrative Genomics Viewer (IGV)[30]. The accession number of the dataset of this study is ERP002413 (Sequence Read Archive – EBI at www.ebi.ac.uk). candidate genes of the region were excluded and LGMD1F remained unsolved for many years. In the last few years, the techniques of next-generation sequencing (NGS) coupled with target enrichment protocols enhanced the molecular genetic diagnostics [14,15]. We studied the original Spanish family with additional family members by exome sequencing [16] using two different NGS platforms. We sequenced the whole exome of four affected individuals and identified a number of new variations, one of which was completely new, shared by all affected subjects, and mapped to 7q32. Methods Ethics Statement This study adhered to the tenets of the Declaration of Helsinki. Subjects for this study were recruited at Padua University and exome analysis was performed at the Second University of Naples and at the Telethon Institute of Genetics and Medicine. Participants were informed of the nature and risks of the study, and signed consent forms were obtained. The institutional review board of the Second University of Napoli (SUN) reviewed and approved this study (prot. AOP-SUN 862). Mutation Detection We designed both cDNA and intronic primers to amplify the cDNA and the 22 coding exons plus the 3’UTR exon of the TNPO3 gene (MIM 610032; NM_012470.3, NM_001191028.2) (Table S2). In addition, we sequenced all the other exons at the disease interval that were inadequately covered (,10x) (Table S3). We also designed additional primers to map the alternatively spliced products. We purified the amplicons and sequenced them by using the fluorescent dideoxyterminator method on an automatic sequencer (ABI 3130XL). Patients Nineteen patients were included in the clinical study, and they all fulfilled the diagnostic criteria for LGMD that include a characteristic pattern of muscular weakness primarily affecting pelvic girdle, assessed according to MRC Scale and a modified Gardner-Medwin & Walton scale for proximal LGMD. Age at onset was assessed as described. We collected blood from 19 patients and 8 healthy relatives. Skeletal muscle biopsy from the deltoid or vastus lateralis was taken from 2 affected individuals. Immunoblotting Analysis For TNPO3 immunoblotting, muscle samples were homogenized in a lyses assay buffer (Urea 8 M, SDS 4%, 125 mM Tris HCl pH 6.8). The samples were separated on sodium dodecyl sulphate –9% polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. After blocking in 10% no fat dry milk in Tween-Tris-Buffered Saline (TTBS-1X) buffer (10 mM TrisHCl, 150 mM NaCl, 0.05% TWEEN 20) for 1h, the membranes were incubated with primary antibodies in TTBS 1X at room temperature for 2 h. The monoclonal antibody, recognizing a recombinant fragment (Human) from near the N terminus of TNPO3, was used in this experiment with a 1:100 dilution (AbcamH). We also used the rabbit monoclonal antibody AntiTNPO3 antibody [EPR5264] (ab109386) that recognizes a synthetic peptide corresponding to residues near the C terminal of Human TNPO3. This was used for WB at 1:300 dilution. Following primary antibody incubation and rinses, the membranes were incubated with the secondary antibody, goat antimouse immunoglobulin conjugated with horseradish (Sigma), with 1:10,000 dilution in 0.5% dry milk and TTBS 1X. After 45 minutes of antibody incubation and five washes with TTBS 1X buffer, the TNPO3 protein band was visualized with a chemiluminescence reagent (Supersignal, WestPico, Pierce) and exposed to X-ray film. To perform this analysis, Coomassie blue staining was used for the evaluation of the myosin protein expression to understand the variations in the levels of the proteins loaded. Exome sequencing and analysis Enrichment was performed by hybridization of shotgun fragment (average size 141 bp) libraries to Agilent SureSelect Human All Exon 50 Mb (Agilent Technologies, Santa Clara, CA, USA) in-solution capture assays. Using the SOLiD system v4 (Life Technologies), we generated an average of 4.2 Gb of mappable sequence data per sample to achieve ,20x mean coverage of the targeted exome. The sequences were analyzed using an automated custom pipeline designed to perform every step of the analysis with the appropriate program or custom script. Sequencing reads were first colour-corrected using SOLiD Accuracy Enhancer Tool (SAET), then mapped to the reference genome (UCSC, hg19 build) using the software BioScope v1.3 (Life Technologies, Carlsbad, CA, USA) and duplicate reads were removed using Picard (http://picard.sourceforge.net). Single nucleotide variations (SNV) and in-del mutation calling analyses were carried out using the diBayes algorithm with medium stringency settings and the SOLiD Small Indel Fragment Tool (www3.appliedbiosystems. com), respectively. One of the samples was sent to a commercial provider (Otogenetics Corporation, Norcross, GA, USA) who performed both whole exome enrichment with the SeqCap EZ Human Exome Library v2.0 (Roche NimbleGen, Inc, Madison, WI, USA) and sequencing with the HiSeq2000 platform (Illumina inc., San Diego, CA, USA). The sequences were analyzed using another automated pipeline designed to handle Illumina data with custom scripts and publicly available software. Paired sequencing reads were aligned to the reference genome (UCSC, hg19 build) using BWA [17] and post-alignment process and duplicate removal was performed using SAMtools [18] and Picard. Further processing PLOS ONE | www.plosone.org Transfection Plasmid pcDNA6/A encoding N-terminal HA-tagged TNPO3 full length was obtained by NR Landau, NewYork University School of Medicine [31]. We subcloned (EcoRI-NheI) HA-TNPO3 exons 1 to 17 in pCS2+ and exons 17 to 22 or 23 were amplified by PCR from cDNA and cloned in pCS2+/HA-TNPO3_1-17 (NheI-XhoI). Four human TNPO3 cDNA constructs were cloned 2 May 2013 | Volume 8 | Issue 5 | e63536 42 May, 2013 TNPO3 Is the LGMD1F Gene Figure 1. LGMD1F family pedigree. Squares represent male; circles represent female; white figures symbolize normal individuals; black figures indicate individuals with clinical muscular dystrophy. The original LGMD1F family has been extended from subject II,2 and now includes 64 LGMD patients of both sexes and five non-penetrant carriers (IV-4, V-26, V-29, V-33, and VI-68). The whole-exome sequencing was performed in four patients indicated by arrows (V-28, VI-36, VI-53, VII-5). doi:10.1371/journal.pone.0063536.g001 synonymous. Considering the dominant mode of inheritance of LGMD1F, we focused on the heterozygous calls and discarded all variants present with a frequency higher than 1% in the NHLBI Exome Variant Server (http://evs.gs.washington.edu/EVS) or 1000genomes [32] large scale projects. The resulting filtered list of 273 variants was composed of 253 missense, 14 stopgain, 2 frameshift deletions, 2 nonframeshift insertions/deletions and 2 stoploss variations. Only two variants were mapped into the disease interval between D7S1822 and D7S2519 (positions: 126,287,140-129,964,025) [11]: a nonsynonymous SNV in the gene IRF5 and a frame-shift deletion that modify the termination codon in the exon 22 (stoploss) in the TNPO3 on chromosome 7q32.1 at position 128,597,310 (GRCh37/hg19). To verify whether we could have missed by NGS other shared variants, we resequenced by the dideoxy-chain termination method all the coding exons and flanking introns of the full 7q32 region with lower/absent coverage (Table S3). No other shared unknown variant was found. In addition, the DNA sample of VI-36 was sent to a commercial provider for exome sequencing using the Illumina platform HiSeq2000. Among 153 variations that were shared by all, the only one in the disease interval was that in the TNPO3 gene (Table 1). Interestingly, this was the only variation of the whole exome that resulted absent in dbSNP137. We also refined the interval: the SNP rs45445295 at the SMO gene at position 128,845,555 was present in some affected members (V-8, VI-60, V-14, VI-11, V-25, V-12), but it was absent in other affected members (VI-57, VI-27, VI-56) and in all non-affected individuals. Therefore, the linked region associated with disease locus was ,1.1 Mb smaller (126,287,140-128,845,555) than that reported by Palenzuela [11]. To confirm the complete co-segregation of the nonstop TNPO3 variant with LGMD1F, we analyzed all available family members, affected and non-affected. We sequenced by the Sanger method all the samples and, in addition, we took advantage of an AluI restriction site that was lost upon mutation. We observed the into the pCS2HA plasmid : 1) Wt TNPO3 isoform with 22 exons; 2) TNPO3 isoform with 22 exons containing del A p.X924C; 3) Wt TNPO3 isoform with 23 exons; 4) TNPO3 isoform with 23 exons containing del A p.X924C. We used 500 ng for transient trasfection of HeLa cells (26105) cells using PolyFect Transfection Reagent (Qiagen) according to manifacturer’s instruction. Cells were grown on glass coverslip put into 12 well plates. They were cultured in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% (v/v) foetal bovine serum and penicillin-streptomycin (GIBCO-Invitrogen) and maintained in a 5% CO2 incubator at 37uC. 48 hours after transfection, cells were fixed with 4% paraformaldehyde in PBS for 10 min at RT, permeabilized in 0,2% Triton X-100 in PBS for 5 min at RT, and blocked for 1 h in Blocking solution (BSA 6%, Horse Serum 5% in PBS). Cells were incubated for 1 h at RT with primary antibodies, followed by 1 h incubation at RT with FITC-conjugated antirabbit and/or Cy3-conjugated anti-mouse antibodies. Results Exome analysis The original LGMD1F family has been extended (Figure 1) to include additional family members in seven generations starting from subject II, 2. The updated pedigree includes 64 LGMD patients of both sexes and five non-penetrant carriers (93% penetrance). To perform an informative exome sequencing analysis, we selected four affected family members (VII-5, VI-53, V-28, and VI-36) with a manifest LGMD phenotype separated by the largest number of meioses. Interestingly, two family members (VI-53 and V-28) were absent from the original family used for the linkage analyses. DNA samples of three individuals (V-28, VI-53, VII-5) were fragmented, enriched using the SureSelect whole exome kit and sequenced by SOliD. DNA, muscle RNA and proteins were extracted for the studies. We found ,20,000 exonic variations for each sample, 5,722 of which were common to all three (Table 1 and Table S1) of which 2,471 were non Table 1. Total and Shared Variants in Patients with LGMD1F. Patient variant type V-28 VI-53 VII-5 Shared by all SOLiD VI-36 Shared by all four 4,212 exonic/splicing 21,105 21,366 17,123 5,722 17,183 non synonymous 11,852 11,713 9,051 2,471 7,831 1,687 heterozygous 9,348 9,138 6,812 644 4,693 153 frequency in EVS and 1000genomes,1% 6,102 5,785 3,860 273 486 10 Within LGMD1F interval 13 11 5 2 1 1 doi:10.1371/journal.pone.0063536.t001 PLOS ONE | www.plosone.org 3 May 2013 | Volume 8 | Issue 5 | e63536 43 May, 2013 TNPO3 Is the LGMD1F Gene Figure 2. Sequence analyses of the TNPO3 mutations. a) Heterozygous delA mutation in Exon 22 of the TNPO3 gene in Proband VII-5. Aligned electropherograms show mutated (top) and wild-type (bottom) sequences; b) Heterozygous. c.G2453A) in exon 21 of the TNPO3 gene; c) Pedigree of the isolated case. doi:10.1371/journal.pone.0063536.g002 complete co-segregation of the TNPO3 variant with the disease (Figure 2a and Table S4). We extended the analysis to additional 64 samples from LGMD1 and isolated LGMD cases, using a next generation sequencing approach. In particular, we performed a custom enrichment of exons of genes involved in muscular dystrophies, including TNPO3. In a single individual, we found a heterozygous G.A transition (c.G2453A) in exon 21 of the TNPO3 gene. This point mutation changes the Arginine in position 818 with a Proline (Figure 2b). This is an extreme conserved residue that is predicted to be damaging by all the used bioinformatic tools (SIFT, PolyPhen, Mutation Taster and LRT). Moreover, the variation is not listed in dbSNP and in the other recently developed databases collecting NGS data (Exome Variant Server and 1,000 genomes database) neither in our internal database of 150 samples whose exomes have been sequenced in our lab. This variation has not been found in the healthy sister (Figure 2c). In addition, this patient bears no other major mutation in other 98 ‘‘muscular-disease’’ genes, but a single heterozygous ANO5 variation (Glu95Lys), without a clear significance. Young adult onset has been observed in this patient, showing a PLOS ONE | www.plosone.org Figure 3. Western blot analysis of skeletal muscle tissue with antibodies to TNPO3. Equal amounts of muscle proteins from a LGMD1F patient and a control were run in each lane (10 mg) on a 9% SDS-polyacrylamide gel and then blotted onto nitrocellulose membrane. In this experiment, we used a monoclonal antibody that recognizes a recombinant fragment (Human) near the N terminus of TNPO3 at a 1:100 dilution. A double band is visible in the patient only. doi:10.1371/journal.pone.0063536.g003 4 May 2013 | Volume 8 | Issue 5 | e63536 44 May, 2013 TNPO3 Is the LGMD1F Gene Figure 4. Indirect immunofluorescence analysis of the wt-hTNPO3 compared with delA p.X924C -hTNPO3. Following transient transfections, HeLa cells were incubated for 48 h with normal DMEM and detected by anti-HA immunofluorescence. Nuclei are stained with DAPI (blue). The endogenous protein is recognized using a rabbit monoclonal anti-TNPO3 antibody (green), while the transfected TNPO3 proteins were HA-tagged (red). a) An accumulation around the nucleus is usually observed using the mutant delA p.X924C -hTNPO3. b) The typical intranuclear staining pattern can be observed in cells transfected with wt-hTNPO3 (in red) or c) in non transfected HeLa cells. doi:10.1371/journal.pone.0063536.g004 characteristic LGMD phenotype. Muscular histopathological data evidenced dystrophic features and, in addition, discrete mitochondrial alterations, with sporadic ragged-red fibers and cytochrome c PLOS ONE | www.plosone.org oxidase negative fibers. Mutations in the mitochondrial DNA were excluded. 5 May 2013 | Volume 8 | Issue 5 | e63536 45 May, 2013 TNPO3 Is the LGMD1F Gene Importance of the TNPO3 mutation nuclear export/import of proteins and in the RNA splicing mechanism, we have two hypotheses: 1) this mutation blocks the nuclear export/import because the longer protein is unable to move to the nucleus, but remains outside the nuclear membrane 2) the mutated protein does not interact with the cargo proteins, causing the block of the nuclear import/export. Our present data indicate that TNPO3 is the gene mutated in LGMD1F. Additional functional studies in model organisms are, however, necessary to understand whether the dominant role of these mutations is due to haploinsufficiency or to a dominantnegative mechanism. This should be possible by the use of antisense morpholino oligos in Danio rerio (Zebrafish) where a single and conserved TNPO3 ortholog is present with 792/923 (86%) amino acid identity (Table S5). Advances in the knowledge of limb-girdle muscular dystrophies have been made in the last few years. With LGMD1F, five different autosomal dominant LGMD genes have been so far recognized. The use of NGS technologies promises a revolution in diagnostics and a more rapid characterization of patients. To analyze the effects of the nonstop mutation on TNPO3 gene products, we first performed the mRNA analysis using skeletal muscle biopsy of a patient compared with a normal control. In both cases, we identified two differently spliced muscular versions of the gene, both including exon 22. Form A that also join exon 22 to exon 23 that is non coding and form B that ends in exon 22. These forms encode the same protein, when the DNA sequence is normal, because the stop codon is in exon 22. However, the LGMD1F mutation eliminates this stop and, for both forms, the muscle protein product is extended by the frame-shift. Form A should be 15 amino acids longer (CSHSCSVPVTQECLF), while form B should contain additional 95 amino acids. We then performed immunoblotting analyses of the skeletal muscle biopsy using the anti TNPO3 antibody. While mutant form A is virtually overlapping with wild-type form A, a mutant form B can be appreciated by western blot analysis of muscle samples as a higher molecular weight band (Figure 3). We generated a construct expressing the WT and del A p.X924C allele. HeLa cells were transfected with either the Wt or the mutant TNPO3. The transfected proteins were distinguished from the endogenous TNPO3 by adding a HA-tag. Figure 4 shows that the WT TNPO3 entered the nucleus, while the mutant was usually around the periphery of the nucleus. Supporting Information Table S1 Exome sequencing data. (DOC) Table S2 Discussion Primers designed for TNPO3 amplification and Sanger sequencing. (DOC) Here, we report the identification of a frame-shift variant del A p.X924C at the TNPO3/Transportin-SR2 gene on chromosome 7q32.1 at position 128,597,310 (GRCh37/hg19) in all patients with limb girdle muscular dystrophy 1F. No other variant was shared by four affected members of the family. The variant modifies the true stop codon and encode for two elongated proteins of 15 and 95 amino acids. Interestingly, one flanking SNP (rs12539741 at 128,596,805) has been identified in association with others in the region as a susceptibility locus for primary biliary cirrhosis [33]. Considering that the affected family members may share a ,2.6 Mb-region on chromosome 7q32, there is the possibility that any other rare heterozygous variant could co-segregate in cis with the true LGMD1F mutation. Thus, we searched in a large collection of patients independent TNPO3 disease-associated variations. We found a missense Arg818Pro in an isolated LGMD case co-segregating with the disease in this family. This variation was predicted as causative by the nature of the change and the conservation. Transportin 3 is a member of the importin b super-family that imports numerous proteins to the nucleus, including serine/ arginine-rich proteins (SR proteins) that control mRNA splicing [34,35]. Transportin 1 (TNPO1), also known as karyopherin b-2, mediates the nuclear import of M9-bearing proteins[36], while TNPO2 (karyopherin b -2B) participates directly in the export of a large proportion of cellular mRNAs[37]. There are two main TNPO3 proteins: variant 1 that is 923 amino acids long and variant 2 composed of 859 amino acids, while a longer variant of the 3’ terminus (hTNRSR1[35]) was probably due to a sequence artifact. The 923-amino acid protein is found in the skeletal muscle, translated from two equivalent messengers that include or not the 3’ noncoding exon. The TNPO3 nonstop allele hindered the nuclear localization of the protein in HeLa cells. Given the role of TNPO3 protein in the Table S3 Exons inadequately covered by NGS exome sequencing and primers designed for Sanger sequencing. (DOC) Table S4 Co-segregation study. (DOC) Table S5 Alignment of Human and Danio r. TNPO3 proteins. (DOC) Table S6 Shared SNVs in the four samples sequenced by NGS. (XLSX) Acknowledgments We thank all the patients and their families for their contribution to this work. We acknowledge the Neuromuscular Bank of Tissues and DNA samples (NMTB) for collecting samples (C.A. and M.F.), Stefania Crispi and Luigi Leone at the IGB Facility of Next Generation Sequencing using the SOLID platform, and Anna Cuomo and Rosalba Erpice for Sanger sequencing. The authors would like to thank the NHLBI GO Exome Sequencing Project and its ongoing studies which produced and provided exome variant calls for comparison: the Lung GO Sequencing Project (HL102923), the WHI Sequencing Project (HL-102924), the Broad GO Sequencing Project (HL-102925), the Seattle GO Sequencing Project (HL102926) and the Heart GO Sequencing Project (HL-103010). We also thank Gopuraja Dharmalingam and the TIGEM Bioinformatics Core for support in exome data analysis and Giuseppina Di Fruscio for the analysis of isolated cases of LGMD and Marina Mora for helpful suggestions. Author Contributions Conceived and designed the experiments: VN MM RR CA. Performed the experiments: AT AG MF MM EP FDVB MS GP LM VN. Analyzed the data: CA VN RR MM. Contributed reagents/materials/analysis tools: AT MF EP GR LM GS CA VN. Wrote the paper: VN. References 1. Nigro V (2003) Molecular bases of autosomal recessive limb-girdle muscular dystrophies. Acta Myol 22: 35–42. 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Pollard VW, Michael WM, Nakielny S, Siomi MC, Wang F, et al. (1996) A novel receptor-mediated nuclear protein import pathway. Cell 86: 985–994. 37. Shamsher MK, Ploski J, Radu A (2002) Karyopherin beta 2B participates in mRNA export from the nucleus. Proc Natl Acad Sci U S A 99: 14195–14199. 7 May 2013 | Volume 8 | Issue 5 | e63536 47 30 April 2013 J Neurol (2013) 260:2033–2041 DOI 10.1007/s00415-013-6931-1 O RI GIN AL COM MUN ICAT ION Clinical phenotype, muscle MRI and muscle pathology of LGMD1F Enrico Peterle • Marina Fanin • Claudio Semplicini Juan Jesus Vilchez Padilla • Vincenzo Nigro • Corrado Angelini • Received: 15 March 2013 / Revised: 11 April 2013 / Accepted: 16 April 2013 / Published online: 30 April 2013 Springer-Verlag Berlin Heidelberg 2013 Abstract Of the seven autosomal dominant genetically distinct forms of LGMD so far described, in only four the causative gene has been identified (LGMD1A-1D). We describe clinical, histopathological and muscle MRI features of a large Italo-Spanish kindred with LGMD1F presenting proximal-limb and axial muscle weakness. We obtained complete clinical data and graded the progression of the disease in 29 patients. Muscle MRI was performed in seven patients. Three muscle biopsies from two patients were investigated. Patients with age at onset in the early teens, had a more severe phenotype with a rapid disease course; adult onset patients presented a slow course. Muscle MRI showed prominent atrophy of lower limb muscles, involving especially the vastus lateralis. Widening the patients population resulted in the identification of previously unreported features, including dysphagia, arachnodactyly and respiratory insufficiency. Muscle biopsies showed diffuse fibre atrophy, which evolved with time, chronic myopathic changes, basophilic cytoplasmic E. Peterle M. Fanin C. Semplicini C. Angelini (&) Department of Neurosciences, University of Padova, Biomedical Campus ‘‘Pietro d’Abano’’, via Giuseppe Orus 2B, 35129 Padova, Italy e-mail: [email protected] J. J. V. Padilla Servicio de Neurologı́a, Hospital Universitario La Fe, Valencia, Spain V. Nigro Department of Pathology, II University of Naples, Naples, Italy V. Nigro Telethon Institute for Genetics and Medicine, Naples, Italy C. Angelini IRCCS San Camillo Hospital, Venice, Italy areas, autophagosomes and accumulation of myofibrillar and cytoskeletal proteins. The LGMD1F is characterized by a selective involvement of limb muscles with respiratory impairment in advanced stages, and by different degrees of clinical progression. Novel clinical features emerged from the investigation of additional patients. Keywords Limb girdle muscular dystrophy LGMD1F Clinical phenotype Muscle MRI Introduction Autosomal dominant limb girdle muscular dystrophies (LGMD type 1) are a heterogeneous group of inherited disorders, which are characterized by progressive involvement and wasting of proximal limb girdle muscles. Currently, eight genetically defined autosomal dominant LGMD subtypes (LGMD1A-1H) have been identified. The diagnosis of LGMD1 might be obtained on the basis of the pattern of inheritance, clinical examination, muscle imaging and muscle biopsy. The causative gene has been so far identified only in four forms, LGMD1A-1D, complicating the distinction between LGMD1 patients on clinical ground and promoting a more in-depth knowledge of clinical, radiological and morphological study. This is a compelling issue in rare forms of LGMD, such as LGMD1F, which has previously been reported [1, 2] in the same large Spanish family with proximal limb and axial muscle weakness we investigated in the present study. Clinical, histological and genetic mapping to 7q32.1-2 have been reported in 32 patients, and the anticipation phenomenon was proposed [1, 2]. The purpose of this paper is to obtain a thorough investigation of this family with LGMD1F by further 123 48 30 April 2013 2034 clinical, radiological and histopathological analysis in an extended number of patients. Methods Patients and neuromuscular examinations The patients investigated in the present study were recruited either during an hospitalization or through outpatient examination, organized by a family association. The clinical data were collected in 24 patients, including seven previously non-investigated cases, by both a complete clinical neuromuscular examination and a standardized clinical questionnaire, whereas in five cases the data were obtained only from the clinical questionnaire. All patients gave informed consent to the participation to a quantitative neuromuscular evaluation according to the approved clinical protocol indicated by Local Regulation. Muscle biopsies and MRI investigation were done after local ethical committee approval and written consent has been obtained. A complete clinical examination was conducted by the same physician to assess muscle atrophy and hypertrophy, gait and posture, presence of joint contractures, scoliosis, scapular winging, individual muscle weakness, difficulty in climbing stairs or in performing Gowers’ manoeuvre, age at loss of independent ambulation. Muscle strength of 18 muscle groups, bilaterally, was assessed using the Medical Research Council (MRC) Scale. The age at onset and the clinical severity of the disease at periodical clinical examinations was graded using a standardized clinical questionnaire which included the modified Gardner-Medwin and Walton (GM-W) scale: grade 1 = normal gait, unable to run freely; grade 2 = tiptoe walking, waddling gait, initial Gowers’ sign; grade 3 = overt muscle weakness, climbing stairs with banister; grade 4 = difficulty rising from a chair; grade 5 = unable to rise from the floor; grade 6 = unable to climb stairs; grade 7 = unable to rise from a chair; grade 8 = unable to walk unassisted; grade 9 = unable to eat, drink or sit without assistance. In 24 patients the respiratory function was evaluated by spirometry in a standing or a sitting position. Muscle morphometry, histopathology and immunohistochemistry We investigated muscle pathology in three biopsies from two female patients (daughter and mother) who underwent diagnostic open biopsies (obtained after written consent) from the left vastus lateralis muscle (case 1, at age 12 and 28 years; case 2, at age 53 years). J Neurol (2013) 260:2033–2041 Muscle samples were snap-frozen in liquid nitrogenchilled isopentane, cross-sectioned and routinely stained to assess the histopathological features. A morphometric study of muscle fibers was conducted on sections stained for haematoxylin-eosin (H&E), which were used to digitalize five to seven non-overlapping random fields, using a 109 microscope objective (Zeiss Axioskop, Gottingen, Germany). Images were captured using a Photometrics CoolSnap camera (Roper Scientific, Ottobrunn, Germany). NIH ImageJ software (v.1.34) was used to trace the borders of 200–500 fibers and calculate fiber cross-sectional area (normal range 708–3846 lm2), fiber diameter (normal range 30–70 lm), coefficient of size variability (normal range 0–250), and fiber atrophy factor and hypertrophy factor, which are the expression of the proportion of abnormally small or large fibers in the biopsy (normal range 0–150) [3]. These two latter parameters have been developed to give different importance to fibers with mild or severe degree of change of fiber size and to detect atrophy or hypertrophy that may not be otherwise apparent by simply calculating the average diameter. Muscle cross sections were processed by immunohistochemistry using a panel of antibodies against desmin (MAB1698, Chemicon, Temecula, CA, USA; 1:50), myotilin (RSO34, Novocastra Laboratories, Newcastle, UK; 1:100), titin (MCN627, YLEM, Avezzano, Italy; 1:50), nebulin (N9891, Sigma Chem. St. Louis, MO, USA; 1:50), alpha-actinin (MCV916, YLEM; 1:50), caveolin-3 (610420, BD Transduction Laboratories, Lexington, KY, USA; 1:50), in order to investigate sarcomeric and myofibrillar and membrane components. For this purpose, 8 lm thick sections were blocked for 15 min with 1 % bovine serum albumin in PBS and incubated for 1 h with primary antibodies. After washes, specific labelling was developed by immunofluorescence, using anti-mouse cyanine-3 conjugated Ig (Caltag, Burlingame CA) diluted 1:100 and incubated for 30 min. Sections were mounted with antifading medium and examined with epifluorescence microscopy. Immunoblotting of MuRF-1 Conventional immunoblot analysis was conducted using muscle sections which were dissolved in Laemmli loading buffer, boiled for 5 min and centrifuged. Proteins were resolved by SDS-PAGE electrophoresis and blotted to nitrocellulose membrane. Blots were air-dried, blocked with 5 % non-fat milk in Tris-Tween-20 saline buffer (TTBS) and incubated overnight with a polyclonal antibody against MuRF-1 (MP3401, ECM Biosciences, Versailles, KY, USA), diluted 1:500 in TTBS. After a thorough washing, the immunoreactive bands were 123 49 30 April 2013 J Neurol (2013) 260:2033–2041 visualized using anti-rabbit peroxidase-conjugated antibodies and the chemioluminescent method (GE Healthcare, UK). The quantity of MuRF-1 protein in the patients’ samples was determined by densitometry using ImageJ software v.1.34 (normalizing the MuRF-1 band on blots to the myosin band in the post-transfer Coomassie blue-stained gels), and was expressed as a percentage of controls. MRI imaging Muscle MRI was performed in seven patients (1 in Padova, 6 in Valencia). A 1.5-T MRI system (Avanto, Siemens, Erlangen, Germany) was used to investigate body segments with axial scans in T1-weighted and turbo inversion recovery magnitude (TIRM) sequences. Patients underwent scans of the scapular girdle, right upper arm and both lower limbs. Fibro-fatty replacement was assessed in T1 spinecho sequences. Areas of signal hyper-intensity were scored as areas of fibro-fatty infiltration, whereas areas of signal hypo-intensity were interpreted as oedema-like changes. The severity of fibro-fatty replacement and its distribution in muscles were scored using the modified Mercuri’s Scale [4]. We used T1 sequences at the thigh level, at about 15 cm from the head of the femur, corresponding to the second slide of MRI in lower extremities, to measure the muscle area of the left quadriceps femoris and vastus lateralis in 1 LGMD1F and ten patients with various neuromuscular diseases, matched by sex and age. The borders of the muscles were outlined on digital MRI images, the area was calculated using MedStation software (v. 4.9) and expressed as mm2. Results Pattern of inheritance We studied a large LGMD1F family of Italian-Spanish origin. In our study, the family pedigree has been reconstructed up to the 7th generation, updating to the most recent generation, and including a novel branch of the family. The pedigree includes now 61 patients (30 females, 31 males) who are clinically affected with LGMD. The pattern of inheritance is clearly autosomal dominant, with high penetrance (94 %), due to some individuals who were reported to be unaffected even if they transmitted the mutant allele to their offspring. The anticipation phenomenon, which has previously been suggested [1], was not confirmed in our series of patients. 2035 Clinical features We collected the clinical data from a total of 29 patients (Table 1; Fig. 1). At onset, the symptoms included difficulty in running or in climbing stairs and weakness and atrophy in the proximal lower limb muscles. The age at onset ranged from 1 to 31 years (mean = 10.2 ± 6.7). Only one patient had onset before 5 years and three after age 20 years. At the time of the clinical study, the patients were aged from 15 to 78 years (mean = 45), and presented a variable degree of impairment of pelvic girdle muscles, with trouble climbing stairs or getting up from the floor. In the more advanced stages of the disease, the weakness involved also the axial and upper girdle muscles, leading to skeletal deformities, such as scapular winging, scoliosis, and joint contractures. One case had drop-head syndrome. A generalized atrophy of muscle mass was a common feature, but the muscles more frequently involved were deltoid and triceps brachii in the upper limbs (Fig. 1), and the quadriceps femoris and the anterior compartment of the leg muscles. Specific clinical pointers and indicators for LGMD1F are skeletal abnormalities such as arachnodactyly (Fig. 1), pes cavus, and mild Achilles tendon retraction. Macroglossia, mild facial weakness, calf hypertrophy gynecomastia and dysarthria were only occasionally observed. Dysphagia was found in 8/29 cases and appeared to be a relatively frequent and previously undescribed clinical feature. Table 1 Clinical features in LGMD1F Characteristics Number of patients Infancy onset \5 years 1/29 Childhood/Juvenile onset \15 years 25/29 Adult onset [20 years 3/29 Early loss ambulation \35 years 3/29 Scapular winging 4/29 Calf hypertrophy 2/29 Respiratory involvement 9/24 Scoliosis 13/24 Arachnodactyly 5/24 Achilles tendon contractures 4/29 Pes Cavus 2/24 Gynecomastia 3/24 Macroglossia 1/24 Dysarthria 1/24 Dysphagia 8/29 Mild facial weakness 1/24 In 24 patients a complete clinical evaluation has been obtained; in five additional patients the clinical data have been obtained by a clinical questionnaire 123 50 30 April 2013 2036 J Neurol (2013) 260:2033–2041 Fig. 1 Pictures from five different LGMD1F patients. Note atrophy of upper girdle muscles, especially deltoid and triceps brachii (a–e), causing difficulty in lifting arms over the head (b) and scoliosis (d). Some patients showed arachnodactyly (b, f), finger contractures (f, h, i) and atrophy of hand muscles (f, g) 123 51 30 April 2013 J Neurol (2013) 260:2033–2041 The natural history or progression of the disease has been reconstructed in 29 patients using the GM-W scale, which evaluates the main motor functions. The Gower’s sign (grade 4) occurred in average at 23 years, the inability in getting up from the floor (grade 5) occurred at 33 years, the inability in getting up from a chair (grade 6) at 41 years (Fig. 2). In some patients the disease course was rapidly progressive, resulting in three patients in early loss of ambulation (grade 8) before age 30 years. Clinical examination of such patients revealed an advanced wasting of lower girdle muscles associated with a severe involvement of the upper girdle muscles, joint contractures, and severe impairment of respiratory function. A rapid worsening of symptoms was reported to have occurred following intense physical exercise (five cases), alcohol intake (one case), long periods of inactivity (two cases), benzodiazepine overuse (one case), and pregnancy (three cases). Another group of patients presented a more slowly progressing course of the disease, leading to loss of ambulation after age 65 years in three cases (Fig. 2). Nine patients (38 %) had moderate/severe respiratory involvement (forced vital capacity below 60 % of normal) that caused also sleep disturbances. Two patients underwent a complete cardiological evaluation (including ECG and echocardiography) that resulted within normal limits, and; therefore, cardiological examination was not pursued in additional cases. Electromyography performed in seven patients showed myopathic changes. Creatine kinase (CK) levels measured in seven cases was either normal or up to threefold increased. 2037 Muscle MRI According to the T1 sequences, the muscle atrophy that resulted was more pronounced in the lower limb muscles than in the upper girdle, affecting mainly the vastus lateralis muscle in the thigh and the triceps surae muscle in the leg (Fig. 3). The fibro-fatty replacement correlated with the degree of muscle atrophy, as observed in other forms of LGMD, i.e., calpainopathy. In case 1, the area of quadriceps femoris and vastus lateralis muscles were 62 and 70 % lower (2,395 and 690 mm2, respectively) than the mean of ten neuromuscular controls (3,843 and 991 mm2, respectively). Muscle histopathology, morphometry, immunohistochemistry, immunoblotting Muscle biopsies obtained from the two patients investigated, showed heterogeneous histopathological features (Fig. 4). Both muscles from case 1 (at age 12 and 28 years) showed a diffuse and progressive muscle fibers atrophy, whereas the muscle from case 2 showed chronic myopathic changes, such as increased fiber size variability, increased central nuclei, nuclear clumps, fiber splitting, endomysial fibrosis, type 1 fibers prevalence. Common features of all three muscle biopsies were basophilic cytoplasmic regions and increased cytoplasmic reaction for lyososomal acid phosphatase even in nondegenerating fibers. In case 2, muscle fiber morphometric analysis (Fig. 5) revealed normal value of fiber diameters but fiber size variability was highly increased because of the presence of Fig. 2 Lineplot describing the clinical course in 29 LGMD1F patients. The clinical functional grade was assessed using the modified Gardner-Medwin & Walton scale. Patients showing a rapid course (wheel-chairbound before age 30 years, grade = 8) are indicated in thick line; patients showing a slower course are indicated in thin line 123 52 30 April 2013 2038 J Neurol (2013) 260:2033–2041 Fig. 3 Muscle MRI T1 sequences from two different patients at the level of the thigh (a, c) and calf (b). Note fibrofatty replacement and atrophy of vastus lateralis in the thigh (a, c) and triceps surae in the leg (b) atrophic and hypertrophic fibers (increased coefficient size variability, cross sectional area, atrophy factor, and hypertrophy factor). Conversely, in case 1, the most relevant change was a generalized fiber atrophy (diameter, atrophy factor, coefficient size variability), which was significantly increased in the second biopsy done at a distance of 13 years, as compared with the first biopsy (p \ 0.001) and with the values in case 2 (p \ 0.001). In both patients, many fibers showed large intracytoplasmic areas with accumulation of cytoskeletal (desmin) or myofibrillar (myotilin, telethonin) proteins (Fig. 4), and large protein aggregates. Immunoblot analysis of MuRF-1 protein, a marker of ubiquitin–proteasome degradation pathway leading to muscle atrophy, showed normal expression level in case 2 (98 % of control mean) but highly increased levels in the second biopsy from case 1 (250 % of control mean) (Fig. 5). Discussion The investigation of a previously reported family with LGMD1F has been expanded by inclusion of additional seven unreported patients, the clinical follow-up of 17 patients, MRI investigation and further muscle histopathological analysis. The widening of the patients population in such a rare form of LGMD, has resulted in the identification of novel clinical features of the disease. In particular, dysphagia was observed in 27 % of cases, arachnodactyly with or without finger contractures was found in 21 % of patients, and dysarthria and calf hypertrophy were occasionally found. Typically, the first symptom was difficulty in climbing stairs. The disease course appeared to be slow and relatively benign in most adult patients. Only three patients have lost ambulation before age 30 years. Muscle 123 53 30 April 2013 J Neurol (2013) 260:2033–2041 Fig. 4 Muscle biopsy from case 1 (28 years) (a–h), and case 2 (54 years) (i–p) stained for H&E (a, b, i), Gomori trichrome (c, j) and acid phosphatase (d, k, l), and immunolabeled with antibodies against desmin (e, f, g, m, n), myotilin (h, o) and caveolin-3 (p). A significant and generalized reduction of fiber size was observed in case 1 (a), whereas in case 2 there were more prominent chronic changes, such 2039 as atrophic angulated fibers, increased central nuclei, fiber size variability and fiber splitting (i, j). In both patients some fibers showed basophilic cytoplasmic areas (b, c, i, j), which were reacting for lyososomal acid phosphatase (d, k, l), and characterized by accumulation of cytoskeletal and myofibrillar proteins (e–h, m–o). Magnification 9100 (a), 9200 (b–e, g, h), 9300 (f, i–p) 123 54 30 April 2013 2040 Fiber diameter A 100 * * * 14000 * * 12000 10000 µ m2 µm Fiber cross sectional area * 75 50 8000 6000 4000 25 2000 0 0 Fiber hypertrophy factor Fiber atrophy factor 1000 1000 750 750 units units Fig. 5 Panel A Histograms showing the mean values of different muscle fiber morphometric parameters (Fiber diameter, Fiber cross sectional area, Fiber atrophy factor, Fiber hypertrophy factor, Coefficient of fibers size variability) observed in the three muscle biopsies from two patients obtained at different ages. Dotted rectangles indicate the range of normal values. In case 1, average fiber diameters were normal but their variability was highly increased because of atrophic and hypertrophic fibers. Conversely, in case 2, the most relevant change was a generalized fiber atrophy, which was significantly increased in the second biopsy (*p \ 0.001). Panel B Immunoblot analysis of MuRF-1 protein in muscle biopsies from controls (Cntr1, Cntr2), and case 1 (28 years) and case 2. After normalization with myosin protein content in the post-transfer Coomassiestained gel, MuRF-1 protein quantity was normal in case 1 (98 % of control mean) and highly increased in case 2 (250 % of control mean) J Neurol (2013) 260:2033–2041 500 500 250 250 0 0 Case 1 (12 years) Coefficient fiber size variability Case 1 (28 years) 800 Case 2 (54 years) units 600 B 400 Case 1 Case 2 Cntr 1 Cntr 2 200 0 involvement occurred mostly in the proximal muscles, but axial muscles were also involved (one case had drop-head syndrome) and clinical features included dysphagia and occasional muscle pain. As compared with LGMD1G [5], that was described in a family with limb weakness associated with a striking limitation of finger and toe flexors (to be considered as important clinical indicators), in this family we frequently observed arachnodactyly and dysphagia.The LGMD1A has also dysarthria, while LGMD1B is characterized by cardiac conduction defects [6, 7], which are absent in LGMD1F. In LGMD1F, muscle MRI demonstrated a proximal involvement of scapular and pelvic girdles. We used a new muscle-imaging quantitative index of the area of quadriceps femoris and vastus lateralis muscles, which, even in the early onset patients, showed a correlation with the degree of muscle fibre atrophy in the same biopsied muscle. The typical clinical symptoms of LGMD1F are progressive muscle atrophy, myopathic EMG, fibro-fatty replacement of muscle on MRI, and active changes in muscle biopsy. The cytoplasmic inclusions and myofibrillar desmin-myotilin positive aggregates in muscle fibers share similarities with the morphological findings observed in myofibrillar myopathies. The changes we observed in biopsies from the quadriceps femoris muscles correspond 123 55 30 April 2013 J Neurol (2013) 260:2033–2041 to the different degree of involvement observed by muscle imaging. Muscle imaging with MRI is increasingly used to determine the patterns of muscle involvement in LGMD. The most consistent example is LGMD2A in which a selective involvement of hip extensors and adductors muscles is observed. In LGMD2J, tibial muscle involvement is frequently found. The muscles from our patients showed protein aggregates and autophagosomes [8], analogous to those seen in other dominant myopathies with protein aggregates, such as myofibrillar myopathies, and also observed in LGMD1D and LGMD1A [6, 9–11]. On the contrary, LGMD1H is characterized by mitochondrial abnormalities in muscle biopsies with ragged-red fibers [12]. Only Gamez et al. [1] reported similar mitochondrial changes in LGMD1F. It is possible that the primary pathogenetic mechanism causes protein aggregation in the cytoplasm and in the nucleus. However, to clarify this hypothesis, further experimental and clinical data are needed. Post-mitotic differentiated skeletal muscle might be uniquely prone to present toxic proteins in an aggregated state. In agreement with these considerations, p62 protein aggregates [8] and MuRF-1 over-protein expression may suggest an involvement of ubiquitin–proteasome and autophagic degradation pathways in this disorder. The adoption of the next-generation sequencing (NGS) strategy in this family resulted in the recent identification of Transportin-3 (TPNO3) as the causative gene of LGMD1F [13]. This novel result is crucial to understand the link between pathogenetic mechanism and clinical features. Acknowledgments The authors wish to thank all the family members who promoted meetings for neuromuscular examination and blood sample collection. This work was supported by grants from the Association Française contre les Myopathies (13859 to MF, 14999 and 16216 to CA) and the Telethon Italy (GTB12001 and GUP10006 to CA and GUP11006 to UN). 2041 References 1. Gamez J, Navarro C, Andreu AL et al (2001) Autosomal dominant limb-girdle muscular dystrophy: a large kindred with evidence for anticipation. Neurology 56:450–454 2. Palenzuela L, Andreu AL, Gamez J et al (2003) A novel autosomal dominant limb-girdle muscular dystrophy (LGMD 1F) maps to 7q32.1-32.2. Neurology 61:404–406 3. Dubowitz V, Sewry CA (2007) In. Muscle biopsy: a practical approach , 3rd edn. Saunders Elsevier, Philadelphia 4. Stramare R, Beltrame V, Dal Borgo R et al (2010) MRI in the assessment of muscular pathology: a comparison between limbgirdle muscular dystrophies, hyaline body myopathies and myotonic dystrophies. Radiol Med 115:585–599 5. Starling A, Kok F, Passos-Bueno MR, Vainzof M, Zatz M (2004) A new form of autosomal dominant limb-girdle muscular dystrophy (LGMD1G) with progressive fingers and toes flexion limitation maps to chromosome 4p21. Eur J Hum Genet 12:1033–1040 6. Hauser MA, Conde CB, Kowaljow V et al (2002) Myotilin mutation found in second pedigree with LGMD1A. Am J Hum Genet 71:1428–1432 7. Van der Kooi AJ, van Meegen M, Ledderhof TM, McNally EM, de Visser M, Bolhuis PA (1997) Genetic localization of a newly recognized autosomal dominant limb-girdle muscular dystrophy with cardiac involvement (LGMD1B) to chromosome 1q11-21. Am J Hum Genet 60:891–895 8. Cenacchi G, Peterle E, Fanin M, Papa V, Salaroli R, Angelini C (2013) Ultrastructural changes in LGMD1F. Neuropathology. doi:10.1111/neup.12003 9. Harms MB, Sommerville RB, Allred P et al (2012) Exome sequencing eveals DNAJB6 mutations in dominantly-inherited myopathy. Ann Neurol 71:407–416 10. Sarparanta J, Jonson PH, Golzio C et al (2012) Mutations affecting the cytoplasmic functions of the co-chaperone DNAJB6 cause limb-girdle muscular dystrophy. Nat Genet 44:450–455 11. Hackman P, Sandell S, Sarparanta J et al (2011) Four new Finnish families with LGMD1D; refinement of the clinical phenotype and the linked 7q36 locus. Neuromusc Disord 21:338–344 12. Bisceglia L, Zoccolella S, Torraco A et al (2010) A new locus on 3p23-p25 for an autosomal-dominant limb-girdle muscular dystrophy, LGMD1H. Eur J Hum Genet 18:636–641 13. Torella A, Fanin M, Mutarelli M, et al (2013) Next-generation sequencing identifies Transportin 3 as the causative gene for LGMD1F. PLoS One (in press) Conflicts of interest The authors declare that they have no conflict of interest. 123 56 March 29th, 2013 doi:10.1093/brain/awt074 Brain 2013: 136; 1508–1517 | 1508 BRAIN A JOURNAL OF NEUROLOGY Limb-girdle muscular dystrophy 1F is caused by a microdeletion in the transportin 3 gene 1 Research Group on Neuromuscular and Mitochondrial Disorders, Vall d’Hebron Institut de Recerca, Universitat Autònoma de Barcelona, Barcelona, 08035, Spain 2 Biomedical Network Research Centre on Rare Diseases (CIBERER), Instituto de Salud Carlos III, Madrid, 28029, Spain 3 Department of Neurology, Columbia University Medical Centre, New York, NY 10032, USA 4 Department of Pathology and Neuropathology, Institute of Biomedical Research of Vigo (IBIV), University Hospital of Vigo (CHUVI), Vigo, 36200, Spain 5 Department of Neurology, Hospital Universitari i Politècnic La Fe, València, 46026, Spain, and Biomedical Network Research Centre on Neurodegenerative Disorders (CIBERNED), Instituto de Salud Carlos III, Madrid, 28029, Spain 6 Neuromuscular Disorders Clinic, Department of Neurology, Hospital Universitari Vall d’Hebron, Institut de Recerca, Universitat Autònoma de Barcelona, Barcelona, 08035, Spain 7 Department of Pathology and Cell Biology, Columbia University Medical Centre, New York, NY 10032, USA 8 Centro Nacional de Análisis Genómico, Barcelona, 08028, Spain 9 Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague, Prague, 12808, Czech Republic * These authors contributed equally to this work. Deceased. # These authors contributed equally to this work. † Correspondence to: Ramon Martı́, PhD, Research Group on Neuromuscular and Mitochondrial Disorders, Vall d’Hebron Institut de Recerca, VHIR, Universitat Autònoma de Barcelona, Passeig Vall d’Hebron, 119-129 08035 Barcelona, Spain E-mail: [email protected] Correspondence may also be addressed to: Michio Hirano, MD, Department of Neurology, Columbia University Medical Centre, 630 West 168th Street, P&S 4-423, New York, NY 10032, USA. E-mail: [email protected] Downloaded from http://brain.oxfordjournals.org/ at Universidad de Valencia on May 13, 2013 Maria J. Melià,1,2,* Akatsuki Kubota,3,* Saida Ortolano,4,* Juan J. Vı́lchez,5 Josep Gámez,6 Kurenai Tanji,7 Eduardo Bonilla,3,7,† Lluı́s Palenzuela,1,2 Israel Fernández-Cadenas,1 Anna Přistoupilová,8,9 Elena Garcı́a-Arumı́,1,2 Antoni L. Andreu,1,2 Carmen Navarro,2,4 Michio Hirano3,# and Ramon Martı́1,2,# In 2001, we reported linkage of an autosomal dominant form of limb-girdle muscular dystrophy, limb-girdle muscular dystrophy 1F, to chromosome 7q32.1-32.2, but the identity of the mutant gene was elusive. Here, using a whole genome sequencing strategy, we identified the causative mutation of limb-girdle muscular dystrophy 1F, a heterozygous single nucleotide deletion (c.2771del) in the termination codon of transportin 3 (TNPO3). This gene is situated within the chromosomal region linked to the disease and encodes a nuclear membrane protein belonging to the importin beta family. TNPO3 transports serine/argininerich proteins into the nucleus, and has been identified as a key factor in the HIV-import process into the nucleus. The mutation is predicted to generate a 15-amino acid extension of the C-terminus of the protein, segregates with the clinical phenotype, and is absent in genomic sequence databases and a set of 4200 control alleles. In skeletal muscle of affected individuals, expression of the mutant messenger RNA and histological abnormalities of nuclei and TNPO3 indicate altered TNPO3 function. Our Received November 5, 2012. Revised January 21, 2013. Accepted February 7, 2013. Advance Access publication March 29, 2013 The Author (2013). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: [email protected] 57 March 29th, 2013 LGMD1F is caused by a TNPO3 mutation Brain 2013: 136; 1508–1517 | 1509 results demonstrate that the TNPO3 mutation is the cause of limb-girdle muscular dystrophy 1F, expand our knowledge of the molecular basis of muscular dystrophies and bolster the importance of defects of nuclear envelope proteins as causes of inherited myopathies. Keywords: limb-girdle muscular dystrophy 1F; LGMD1F; TNPO3; transportin 3; c.2771del mutation Abbreviation: LGMD = limb-girdle muscular dystrophy Introduction Materials and methods Patients The reported genealogical investigation of the LGMD1F family (Gamez et al., 2001) disclosed a common ancestor born in south-eastern Spain two generations before the oldest living members. The largest branch originated from Subject II-3 (Gamez et al., 2001) and includes 32 patients with LGMD1F, of whom 28 have been closely followed in our centre (University Hospital La Fe, València, Spain). Functional activity was assessed using the Brooke score (from 1: normal; to 6: no function for upper extremity) (Brooke et al., 1981) and the Vignos score (1: able to climb stairs without help; to 10: bedridden for lower limb function) (Vignos et al., 1963). Muscle strength was graded using the Modified Medical Research Council (MMRC) scale. Whole-body muscle imaging was performed on a 1.5 T or 3 T MRI scanner. Abnormal muscle signal intensity was ranked according to Mercuri scale (Mercuri et al., 2002): 1, normal appearance; 2, motheaten appearance with scattered small areas of increased signal involving 530% of muscle volume; 3, moderate involvement (a late motheaten appearance with numerous discrete areas of increased signal with incipient confluence, involving 30–60% of muscle volume); 4, severe involvement (washed-out or fuzzy appearance due to confluent areas of increased signal, or complete muscle replacement by connective tissue and fat with only a rim of fascia and neurovascular structures). All pedigree identifiers in this report refer to the updated family tree shown in Supplementary Fig. 1, unless otherwise indicated. Downloaded from http://brain.oxfordjournals.org/ at Universidad de Valencia on May 13, 2013 The limb-girdle muscular dystrophies (LGMDs) comprise a group of genetically heterogeneous disorders characterized by progressive and predominantly proximal muscle weakness with histological signs of degeneration and regeneration in muscle (Bushby, 2009). As a result of molecular characterization and improved clinical criteria, the classification and nomenclature of LGMDs have evolved over the last two decades. The canonical categorization of LGMD into autosomal dominant-LGMD (LGMD1) and autosomal recessive (LGMD2) forms is being refined by a classification based on the affected proteins and their correspondent genes (Nigro et al., 2011) In 2001, we reported the clinical and morphological phenotype of a novel form of autosomal dominant-LGMD affecting 32 individuals in a large Spanish kindred spanning five generations (Gamez et al., 2001). Clinically, the disorder was characterized by muscle weakness primarily affecting the pelvic and shoulder girdles with a wide variability in the age at onset (1–58 years old), disease progression rate and severity. The disease generally ran a benign clinical course, but some individuals with childhood or juvenile onset manifested severe widespread myopathy leading to wheelchair dependency and respiratory insufficiency. Additional clinical features of LGMD1F as well as more detailed descriptions of its time-course and pattern of muscle involvement are presented here. Initially, the presence of rimmed vacuoles and filamentous inclusions in myofibres of affected subjects prompted us to consider a diagnosis of hereditary inclusion body myopathy (Huizing and Krasnewich, 2009). Hereditary inclusion body myopathy typically presents as an autosomal recessive trait and is due to mutations in GNE (9p13.3; Genebank NM_005476); however, a few cases of autosomal dominant hereditary inclusion body myopathy have also been described and linked to chromosome 17p13.1 (Martinsson et al., 1999) (IBM3 OMIM #605637) or to 7q22.131.1 (Lu et al., 2012) (IBM4). Hereditary inclusion body myopathy was ruled out in our family because initial genetic analysis using simple sequence repeat markers indicated that the disorder was not linked to hereditary inclusion body myopathy. Our subsequent studies using genome-wide markers demonstrated a novel locus for this autosomal dominant-LGMD at the chromosomal locus 7q32.1-32.2, between markers D7S1822 and D7S2519, containing 66 genes. These data confirmed that this family has a genetically distinct form of autosomal dominant-LGMD that was classified as LGMD1F (Palenzuela et al., 2003) (OMIM #608423). However, the identity of the mutant gene has been elusive so far, despite attempts to find it following different strategies. Here, using a whole genome sequencing approach, we have identified the causative mutation of the LGMD1F, a single nucleotide deletion in the termination codon of transportin 3 (TNPO3). The histochemical and ultrastructural findings, together with the molecular results at DNA, RNA and protein levels, fully support the pathogenic role of this mutation in LGMD1F. Muscle biopsies Muscle biopsies from the deltoid or vastus lateralis from 5 of the 32 affected individuals of the family had been performed in the years 1993 and 1994 under informed consent (Gamez et al., 2001) and stored at 80 C at the Neurological Tissue Biobank of Vigo University Hospital. Frozen muscle specimens from Subjects IV-6, IV11 and IV-21 (Supplementary Fig. 1) (Gamez et al., 2001) were retrieved from the Biobank and further studied by light microscopy. For ultrastructural studies, original electron microscopy micrographs were re-examined. Stored Epon-embedded blocks were used to obtain new ultrathin sections, and were studied under a Philips 58 March 29th, 2013 1510 | Brain 2013: 136; 1508–1517 CM100 transmission electron microscope equipped with a digital camera and the ITEM SYSTEM VELETA (FEI Company) software. DNA and RNA isolation and complementary DNA synthesis DNA was extracted from anticoagulated blood from affected and unaffected individuals using a standard phenol–chloroform method. RNA was extracted from skeletal muscle biopsies from Subjects IV-6 and IV-21 and one unrelated healthy control using the RNeasy Kit (QIAGEN), and treated with the deoxyribonuclease I, amplification grade (Invitrogen) to eliminate any traces of DNA. Then, complementary DNA was synthesized using the High Capacity complementary DNA Reverse Transcription kit, which uses random hexamers (Applied Biosystems). Sequencing libraries were constructed according to the TruSeqTM DNA sample preparation protocol (Illumina) with minor modifications, in particular the double size selection. Two micrograms of genomic DNA were fragmented with a Covaris E210 and size selected to 300–700 bp. Resulting fragments were end-repaired, adenylated, ligated to Illumina paired-end adaptors and size selected to very tight sizes using an E-Gel (Life Technologies). Size selected adapter-insert fragments (two insert sizes: 430 bp and 460 bp) were amplified with 10 PCR cycles and sequenced on an Illumina HiSeq 2000 platform with paired end run of 2 100 bp. Base calling and quality control was performed on the Illumina RTA sequence analysis pipeline. Sequence reads were trimmed to the first base with a quality over 30 and mapped to Human genome build hg19 (GRCh37) using GEM mapper (Marco-Sola et al., 2012), allowing up to four mismatches. Reads not mapped by GEM mapper (4%) were submitted to a last round of mapping with BFAST (Homer et al., 2009). Results were merged and only uniquely mapping non-duplicate read pairs were used for further analyses. SAM tools suite version 0.1.18 (Li et al., 2009) with default settings was used to call single nucleotide variants and short indels. Variants on regions with low mappability (Derrien et al., 2012), with read depth 510 or with strand bias P-value 5 0.001 were filtered out. The population frequency of the variants was assessed by comparing to several databases: the 1000 Genomes Project (http://www.1000genomes.org/), NHLBI Exome Sequencing Project (ESP) release ESP5400 (http://evs.gs.washington. edu/EVS/), and our internal database of sequence variants identified in a set of 4100 control samples). The effect prediction was performed with Annovar version 2011 Dec20 (Wang et al., 2010) and snpEff version 2.0.5d (Cingolani et al., 2012). Dideoxy-DNA sequencing DNA extracted from blood was used to confirm the segregation of the genotype and the phenotype. A 856 bp fragment encompassing the last coding sequence of the TNPO3 gene was PCR-amplified (forward, 5’-TCCTCAGTCAAGGACCAACCTACCT-3’; reverse, 5’-TCCTGTAAG GGCCAAGCATCCCT-3’), and the product was purified (ExoSAP-IT , Affimetrix) and sequenced using the dideoxy method (BigDye Terminator v3.1 Cycle Sequencing kit, Applied Biosystems). In order to analyse the sequences of RNA species, complementary DNA obtained from skeletal muscle of affected and unaffected individuals was PCR-amplified (644 bp fragment, exons 20–24, primers forward 5’-TCTACTACCCTGGACCACCG-3’ and reverse 5’-GCGCTGATTTTCCCTCACAC-3’) and the resulting fragments were sequenced. Polymerase chain reaction–restriction fragment length polymorphism analysis A 629 bp fragment was PCR-amplified (Forward primer 5’-TCTACTACCCTGGACCACCG-3’ and Reverse primer 5’-CACACCCCCAAACAGGAACT-3’) from skeletal muscle complementary DNA from Subjects IV-6, IV-21 and one unrelated healthy control subject. The products were digested with the restriction enzyme SfaNI (New England Biolabs). The wild-type sequence of the 629 bp amplicon contains a single SfaNI target generating two fragments: 617 bp + 12 bp. The c.2771del mutation generates an additional target, producing the expected restriction fragment pattern of 400 bp + 216 bp + 12 bp. The fragments were resolved by electrophoresis in a 2% agarose gel, visualized by ethidium bromide staining, and the bands were quantitated by densitometry using ImageJ software. Western blot Frozen muscle biopsy samples were homogenized in lysis buffer containing 0.25% NP-40 with protease inhibitor cocktail (cOmplete Mini , Roche Diagnostic), and after centrifugation, supernatants were collected. Concentrations of protein in the supernatants were measured by bicinchoninic acid assay. Aliquots containing 40 mg protein were separated by SDS-PAGE and transferred to a membrane. After blocking with PBS containing 0.5% skimmed milk, the membrane was incubated at 4 C overnight with primary antibodies: antiTNPO3 antibody (ab54353, Abcam 1:50) and anti-beta-actin antibody (20536-1-AP, Proteintech, 1:1000). The immunoprobed membrane was washed with PBS containing 0.5% Tween 20 three times, and was incubated for 1 h at room temperature with peroxidase-conjugated anti-mouse IgG antibody or anti-rabbit IgG antibody. After incubation with secondary antibodies, the membrane was washed with PBS containing 0.5% Tween 20 three times, and was developed with ECL Prime Western Blotting Detection Reagents (GE Healthcare). The membrane was imaged with G:BOX Chemi IR6 (SYNGENE). Downloaded from http://brain.oxfordjournals.org/ at Universidad de Valencia on May 13, 2013 Whole genome sequencing M. J. Melià et al. Anti-TNPO3 and 4’,6-diamidino-2phenylindole staining Six-micrometre thick sections of frozen muscle were fixed in ice-cold acetone for 10 min, incubated for 1 h with 1% bovine serum albumin, and stained at 4 C for overnight with murine antiTNPO3 antibody (ab54353, Abcam) at a concentration of 5 mg/ml. Specimens were then incubated for 1 h with sheep biotinylated antimouse IgG antibody (RPN1001, GE Healthcare, 1:100) followed by 1 h with streptavidin-fluorescein (RPN1232, GE Healthcare, 1:250), mounted, and stained with 4’,6’-diamidino-2-phenylindole dihydrochloride (DAPI) using VECTASHIELD Mounting Medium with DAPI (Vector Laboratories). The stained sections were examined with a confocal microscope (Leica TCS SP5 II , Leica microsystems), and images were obtained with LAS AF (Leica microsystems). 59 March 29th, 2013 LGMD1F is caused by a TNPO3 mutation Results Clinical assessments | 1511 stage 8 at the fifth and seventh decade. Three additional patients reached Vigno stages 6 and 7 in their forties through sixties. Two patients died suddenly at age 57 and 78 due to causes unrelated to the myopathy. Laboratory investigations provided similar results to those previously reported (Gamez et al., 2001); however, electromyography in the current series frequently showed spontaneous activity, and, in 3 out of 8 patients, displayed clear motor neurogenic features. In two patients who presented with intense fatigue, ptosis and transient dysphagia, repetitive stimulation tests and single-fibre electromyography disclosed no abnormalities in neuromuscular transmission. Serum creatine kinase levels were also consistent with the previous report: 40% of the cases showed elevated creatine kinase levels (4500 U/l, maximum 2200). No correlation between clinical severity and creatine kinase levels was identified. Muscle MRI demonstrated variable involvement of scapular and pelvic-femoral muscles, as well as lower leg muscles (Supplementary Fig. 3). A characteristic relationship between muscle MRI abnormalities and degree of impairment was observed with intensity of the MRI signal changes correlating well with the severity of the clinical involvement. In general, scapular-humeral girdle muscles were much better preserved than pelvic-femoral and leg muscles. The percentage of cases with moderate (3) or severe (4) Mercuri scores by muscle group are: (i) scapular girdle: teres major (80%), pectoral (64%), infraspinatus and serratus anterior (55%), deltoids (46%); (ii) lumbar: paraspinal (90%), abdominal oblique (55%) and rectus abdominus (55%); (iii) thigh: sartorius (100%), vastus lateralis, intermedius and medialis (73%), biceps femoris and semitendinosus (55%); and (iv) lower leg: peroneal (91%), gastrocnemius (91%), soleus (73%) and tibialis anterior (70%). Correlations between the clinical severity and the degree of MRI muscle affectation are presented in Supplementary Fig. 3. Subject V-9 represents a mildly symptomatic subject without overt clinical weakness but with Mercury stage 3 abnormalities in lumbar paraspinal, sartorius and peronei muscles. Subject V-7 represents a moderately affected subject (Vignos scale 5) showing a widespread muscle involvement (Mercuri stage 3) of paravertebral and abdominal lumbar muscles, anterior and posterior thighs and diffuse lower leg muscles. Finally, Subject IV-26 corresponds to a severely affected patient (Vignos rating of 7) manifesting Mercuri 3 and 4 grade abnormalties in scapular, lumbar, thigh and lower leg muscles. Downloaded from http://brain.oxfordjournals.org/ at Universidad de Valencia on May 13, 2013 The cohort reported here comprises 30 individuals spanning Generations III to VI; 13 were included in a previous study (Gamez et al., 2001). They presented with limb-girdle and distal muscle weakness with variable distribution, severity, and rate of progression (Supplementary Table 1). Based on age-at-onset, a predominant group of juvenile-onset (onset before age 15) was delineated from an adult-onset form starting in the third and fourth decades (Gamez et al., 2001). Although a similar distribution was observed in the present cohort, the difference between juvenile- and adult-onset groups was not pronounced. Six patients in the fifth and sixth generations had infantile onset disease characterized by a mild delay in motor skill (independent walking was never delayed beyond age 22 months), followed by difficulty rising from the floor to a standing position and climbing stairs without the aid of hand rails. Running and jumping were difficult or impossible. Some manifested an abnormal gait with a mixture of waddling and distal leg weakness. When walking or attempting to stand on heels, patients also demonstrated a peculiar posture of the feet with elevation of the big toe, foot drop and weight-bearing on the lateral soles (Supplementary Fig. 2). In addition, all patients had thin legs and thenar muscle atrophy. Two patients presented early contractures at the heels, knees, and elbows with rigid spine and scoliosis reminiscent of EmeryDreifuss muscular dystrophy, but lacking cardiac involvement. Common initial symptoms in the late-childhood and adolescent group were difficulty running and playing sports. Overt symptoms of pelvic-femoral weakness, such as difficulty in rising from the floor or climbing steps, were also frequent. One patient had exercise intolerance, myalgia and fatigue reminiscent of a metabolic myopathy. Symptoms of pelvic-girdle weakness were the most common presentation in late-onset patients. Weakness and atrophy of shoulder girdle muscles appeared in only 70% of cases, always in later or advanced stages of the disease, and usually showing less degree of involvement than muscles of pelvic-femoral and axial territories. While both mild (Brook scale 1–2) and severe (Brook scale 3–4) cases showed minor scapular winging (Supplementary Fig. 2), prominent scapula alata was never observed. Distal muscle involvement was more frequent (at least 24 of 30 patients) than previously reported. In hands, thenar muscle atrophy was observed in all 24 carefully assessed patients and a high proportion of cases reported difficulty grasping a pencil or opening jars. In lower leg, subclinical muscle weakness was often revealed by asking patients to stand on their heels. Other symptoms associated with the disease were: mild ptosis (five cases), transient dysphagia (nine cases) and episodic vertigo and ataxia (eight cases). Three cases had respiratory muscle involvement; all required non-invasive nocturnal ventilatory support. The course of the disease was highly variable. The two most severe cases with an Emery-Dreifuss-like phenotype were wheelchair-bound in the third decade. Two other cases reached Vigno Brain 2013: 136; 1508–1517 Histological studies Previous analyses of muscle biopsies from five people affected with LGMD1F had revealed increased variability of fibre size and shape, increased endo- and peri-mysial connective tissue, scattered degenerating fibres, occasional central nuclei, abnormal intermyofibrillar network with abnormal Z bands, rimmed vacuoles and abnormally increased mitochondria with rare paracrystalline inclusions (Gamez et al., 2001). These histological features are similar to those recently reported in the same family (Cenacchi et al., 2012). New analyses of muscle biopsies from seven affected patients (Supplementary Fig. 1) confirmed the described abnormalities in myofibres and connective tissue. In addition, we observed unusually enlarged nuclei with central pallor (Fig. 1). These nuclear 60 March 29th, 2013 1512 | Brain 2013: 136; 1508–1517 M. J. Melià et al. Subject IV-6. In A, abnormal myonuclei with an ‘empty’ appearance (arrows) (630). Scale bar = 20 mm. In B, note three abnormal nuclei (arrows) within a myofibre at higher magnification (1000). Scale bar = 10 mm. (C and D) Electron micrographs showing non-branching tubular filaments 18–20 nm in diameter within a muscle fibre (Subject IV-6). Note myelin and membranous bodies surrounding filaments (C), which are characteristic of rimmed vacuoles. Original magnifications: 21 000 and 35 000. Scale bars = 0.5 mm. abnormalities were identified in all seven biopsies in 11.0–25.8% of muscle fibres (Subject III-14: 18.8%; Subject IV-6: 16.7%; Subject IV-11: 14.8%; Subject IV-18: 16.0%; Subject IV-21: 17.4%; Subject IV-36: 25.8%; Subject V-14: 11.0%). These percentages were derived from counts of the number of affected nuclei and the total number of fibres within each biopsy in haematoxylin–eosin stained slides, using a histometric program (Leica Application Suite v 3.8.0). We have not observed these nuclear abnormalities in other myopathies including Duchenne muscular dystrophy, sarcoglycanopathies, Emery-Dreifuss muscular dystrophy due to emerin or lamin A/C mutations, or FHL1 dystrophy. Ultrastructurally, filamentous inclusions, 18 to 20 nm in diameter, were detected within nuclei or in the cytosol of a minority of fibres in two out of five biopsies (Subjects IV-6 and IV-21, Fig. 1). In three biopsies, corresponding to Subjects IV-6, IV-18 and IV-21, light microscopy showed rimmed vacuoles and electron microscopy revealed autophagic vacuoles with prominent pseudomyelin structures, membranous whorls and dense bodies. Immunocytochemical stains for desmin, dystrophin, sarcoglycans, tau, ubiquitin, and amyloid-b proteins, did not show significant alteration or accumulation in muscle fibres. Genetic and molecular studies Initial strategies to identify the genetic cause of the disease included sequencing of candidate genes. Among them, FLNC, encoding filamin c, was extensively investigated because mutations in this gene cause autosomal dominant myofibrillar myopathy (Vorgerd et al., 2005) (OMIM #609524). Studies included dideoxy-DNA sequencing of FLNC exons, flanking introns and promoter regions, Southern and northern blot analyses, and immunohistochemical staining and western blot analyses of muscle biopsies with anti-FLNC antibodies, and revealed normal results compared to controls (data not shown) thereby excluding FLNC as the causative gene. Dideoxy sequencing of 65 additional genes within the region failed to reveal potentially pathogenic mutations, although the presence of heterozygous changes was difficult to be completely ruled out in some of the electropherograms due to suboptimal quality. Comparative Genomic Hybridization (CGH, NimbleGen Systems Inc.) across the linked region excluded genome copy number variations. Segmentation values across the chromosome 7 regions of interest and other chromosomes showed no differences in DNA from two control subjects and two affected individuals, thereby excluding DNA copy number alterations as the cause of the disease (data not shown). Because studies at the DNA level were unrevealing, we performed analyses of messenger RNA levels. Expression of 36 genes included in the critical region was analysed in RNA extracts from skeletal muscle of affected members of the family and unaffected unrelated subjects, using a TaqMan Custom Array 384well microfluidic card (Supplementary Fig. 4 and Supplementary Table 2). No significant differences could be detected in the expression of the genes analysed, except for a moderate increase of Downloaded from http://brain.oxfordjournals.org/ at Universidad de Valencia on May 13, 2013 Figure 1 Light and electron microscopy findings in muscle biopsies. (A and B) Haematoxylin and eosin staining muscle from affected 61 March 29th, 2013 LGMD1F is caused by a TNPO3 mutation | 1513 DNA revealed the coexistence of both mutated and wild-type transcripts in similar amounts, according to the sizes of the peaks of the two overlapped sequences observed in the electropherograms (Fig. 2). This result was confirmed by PCR-restriction fragment length polymorphism analysis, which indicated that 61– 64% of the TNPO3 messenger RNA of the two affected individuals contained the mutant form (Fig. 2). Retrospective review of the real-time PCR results obtained in the microfluidic cards (Supplementary Fig. 4) confirmed that TNPO3 was expressed in skeletal muscle of affected and non-affected persons at similar levels. Taken together, these results demonstrate that the mutated messenger RNA is stable and does not undergo RNA decay. We performed western blot analysis of biopsied muscle samples from four affected subjects and two unaffected control subjects, to assess changes in amount and molecular weight of TNPO3 protein (Fig. 2). The TNPO3 mutation in the family disrupts the termination codon, and is predicted to extend the C-terminus of TNPO3 by 15 amino acids. Using an anti-TNPO3 antibody that recognizes an N-terminus epitope, present in both normal and mutant TNPO3, western blot analysis showed a single band at the same level in muscle from control subjects and affected individuals, and no extra bands were observed in muscles from affected subjects. However, the 15 amino acid size difference between normal and mutant TNPO3 is likely insufficient to distinguish the two proteins by western blot. Relative to control subject muscles, the amount of muscle TNPO3 normalized to beta-actin was increased in one affected subject (Subject IV-36), and decreased in the other three (Subjects V-14, III-14 and IV-18); therefore, there were no significant difference in TNPO3 quantity in mutant versus normal tissue. To assess the effects of the TNPO3 mutation on TNPO3 cellular localization, we performed immunohistochemistry of muscle tissue with anti-TNPO3 antibody. Control muscle stained with antiTNPO3 antibody showed clear nuclear staining and that colocalized with DAPI (Fig. 3I). In muscle of affected individuals, TNPO3 immunostaining was also observed within nuclei, but was unevenly distributed and often limited to the periphery of nuclei, (Fig. 3C and F). Discussion Downloaded from http://brain.oxfordjournals.org/ at Universidad de Valencia on May 13, 2013 NRF1 transcripts and a pronounced increase of LEP transcripts, in skeletal muscles from affected persons. Because LEP, encoding leptin, is highly expressed in adipose tissue (Meier and Gressner, 2004), we suspected that elevated LEP expression reflected fat replacement of affected muscle, rather than a primary pathogenic alteration. We analysed the expression levels of ADIPOQ, which is expressed exclusively in adipose tissue (Maeda et al., 1996), and also found increased levels of this marker of fat tissue in muscles from affected subjects (Relative quantification (RQ) median; interval: 73.8; 23.9–339.0; n = 5) as compared with levels in unaffected subjects (RQ median; interval: 3.0; undetectable -10.3; n = 6). The levels of ADIPOQ transcripts closely correlated with values for LEP messenger RNA (P 4 0.001; Spearman correlation coefficient R = 0.991), thus supporting the notion that increases in LEP transcript reflected fatty replacement of muscle. After our initial efforts to find the genetic cause of LGMD1F failed, we applied a more powerful strategy, whole genome sequencing analysis of DNA from one affected individual (Subject III-12, Supplementary Fig. 1). After intersecting the results of whole genome sequencing with the results from previous linkage analysis (Palenzuela et al., 2003) (chromosome 7: 126 287 120–129 963 917), 3888 variants (3125 single nucleotide variants and 763 indels) were identified, from which 718 were novel, not present in the dbSNP database, build 135 (http://www.ncbi.nlm. nih.gov/projects/SNP/). Additional criteria based on the dominant inheritance of the disease, the population frequency of the variants and effect prediction (see ‘Materials and methods’ section), allowed us to rule out all but one of these variants, a heterozygous mutation in the termination codon of the TNPO3 gene, encoding transportin 3, a protein involved in the translocation of proteins from the cytoplasm to the nucleus (Brass et al., 2008; Cribier et al., 2011). The mutation (c.2771del, reference sequence GeneBank NM_012470.3, Fig. 2) is a single adenine nucleotide deletion in the TAG stop codon, common to the two protein isoforms encoded by the gene. The del-A results in conversion of TAG to TGC codon, encoding cysteine, and extension of the reading frame by 15 codons to a downstream of the termination-signal within the transcript. Thus, the frameshift leads to the predicted mutated TNPO3 protein with 15 additional amino acids at the C-terminus [p.(*924Cysext*15) for isoform 1]. The retrospective analysis of the sequences of this gene revealed that this heterozygous mutation had been missed in the past due to the poor quality of the electropherograms. Then, we performed new dideoxy sequence analysis of TNPO3, which demonstrated presence of c.2771del in each of the 29 clinically affected individuals and absence of the mutation in all 20 clinically unaffected relatives tested. Thus, the sequence data indicate that the mutation segregates with the linked chromosome 7q32.1-32.2 region (Palenzuela et al., 2003) and with the phenotype (Supplementary Fig. 1). To investigate whether the mutated messenger RNA was expressed in skeletal muscle of the affected individuals, complementary DNA was generated using RNA from two skeletal muscle samples (Subjects IV-6 and IV-21, Supplementary Fig. 1), and the 3’-end fragment containing the native stop codon (common to the 3 transcripts described for the gene, TNPO3 gene entry in the NCBI, http://www.ncbi.nlm.nih.gov/gene/23534) was PCRamplified. Sequence analysis of this amplified complementary Brain 2013: 136; 1508–1517 LGMD1F is one of the nine autosomal dominant forms of LGMD. Causative genes had been identified for only five forms of autosomal dominant-LGMD: MYOT (LGMD1A, OMIM #159000), LMNA (LGMD1B, OMIM #159001), CAV3 (LGMD1C, OMIM #607801), DES (LGMD1D, OMIM *125660), and DNAJB6 (LGMD1E, OMIM #603511) (Bushby, 2009; Sarparanta et al., 2012) (see GeneReviewsTM LGMD Overview). In general, these disorders are characterized by adult-onset and milder clinical phenotypes than LGMD2. Although, most individuals harbouring mutations in these genes fulfil the diagnostic criteria for LGMD, some manifest a wider spectrum of clinical phenotypes. The extreme example is LMNA mutations, which have been associated with a broad spectrum of clinical conditions including Dunnigan lipodystrophy, autosomal dominant Emery-Dreifuss muscular dystrophy, cardiomyopathy, Charcot–Marie–Tooth disease and 62 March 29th, 2013 1514 | Brain 2013: 136; 1508–1517 M. J. Melià et al. Downloaded from http://brain.oxfordjournals.org/ at Universidad de Valencia on May 13, 2013 Figure 2 Effects of the c.2771del mutation on TNPO3 messenger RNA and protein. (A) The 3’-terminal coding and untranslated region (UTR) sequences of TNPO3 transcripts, including the 3’-end of the exon 23 (black font) and the 5’-end of the non-coding exon 24 (blue font) of both wild-type and mutant (c.2771del) complementary DNAs. The fragments shown are identical in both transcript variants 1 and 2. The deleted 2771A is labelled with an asterisk. The encoded amino acids are indicated in one-letter code. Changes resulting from the frame-shifted codons in the mutated sequence are indicated in red case, which highlight the disruption of the native TAG stop codon, with a modified C-terminus containing 15 extra amino acids p.(*924Cysext*15) relative to normal isoform 1. (B) Electropherograms showing the complementary DNA sequences from muscle messenger RNA obtained from a healthy control (top, wild-type sequence), and the affected subject IV-6 (bottom) showing the coexistence of both wild-type and c.2771del mutated transcripts at similar amounts. Sequences encompass two different exons (exon 23 in black and exon 24 in blue). A similar result was obtained for the Subject IV-21 (data not shown). (C) PCR-restriction fragment length polymorphism analysis of complementary DNA obtained from skeletal muscle TNPO3 messenger RNA. A 629 bp fragment was PCR-amplified from skeletal muscle complementary DNA from Subjects IV-6, IV-21 and one unrelated healthy control. The products were digested with the restriction enzyme SfaNI. The wild-type sequence of the 629 bp amplicon contains one SfaNI site generating two fragments: 617 bp + 12 bp. Because the c.2771del mutation generates an additional SfaNI site, restriction enzyme digestion produces three fragments: 400 bp + 216 bp + 12 bp. Densitometric analysis of the bands showed that the mutated messenger RNA was 64% (Subject IV-6) and 61% (Subject IV-21) of total TNPO3 messenger RNA. (D) Western blot of muscle specimens from affected subjects and controls. Muscle specimens from four subjects with LGMD1F and two control subjects were analysed by western blot. The anti-TNPO3 antibody showed a clear band at approximately 100 kDa in each lane. No differences in the position of bands and no extra bands were observed. There were no significant differences in the amounts of TNPO3 normalized to beta-actin between affected subjects and controls. 63 March 29th, 2013 LGMD1F is caused by a TNPO3 mutation Brain 2013: 136; 1508–1517 | 1515 affected individuals (Subject IV-36: A–C, Subject V-14: D–F) and two control subjects (one not shown) (G–I) were observed under a confocal microscope. Each specimen was stained both with anti-TNPO3 antibody (A, D and G) and by DAPI (B, E and H), and merged images were generated (C, F and I). TNPO3 staining colocalized with DAPI in control subjects (I). In affected individuals, signals of TNPO3 were also observed within nuclei, but were unevenly distributed (C and F). Scale bar = 40 mm. Hutchinson-Gilford progeria (Worman et al., 2009; Bertrand et al., 2011). Several lines of evidence strongly support pathogenicity of the TNPO3 mutation in this family with autosomal dominant-LGMD: (i) TNPO3 resides within the chromosome 7q32.1-32.2 locus for LGMD1F; (ii) the mutation segregates with the phenotype; (iii) the microdeletion is absent in publicly available genomic sequence databases (dbSNP build 135, 1000 Genomes Project and 5400 NHLBI exomes) and in our set of 4200 Spanish control alleles indicating that all control individuals harbour the canonical TNPO3 TAG termination codon in homozygosity at the position 128 597 311 of the chromosome 7; (iv) the mutation in the termination codon of TNPO3 is predicted to extend the coding sequence at the 3’-end of the messenger RNA and to generate an aberrant protein; (v) the mutated messenger RNA is expressed in the muscle of the affected individuals; and (vi) the detection of histologically abnormal muscle nuclei with atypical nuclear filaments, anomalous TNPO3 immunoreactivity and irregular membranes. These morphological changes of myocyte nuclei indicate that the TNPO3 c.2771del mutation alters nuclear functions, which is consistent with the putative role of the TNPO3 in transport of proteins across the nuclear membrane. We observed similar levels of TNPO3 transcript in skeletal muscle of three healthy control subjects and in five affected subjects (Supplementary Fig. 4). It is likely that the mutant protein, which is predicted to contain 15 additional amino acids at the C-terminus, is expressed in skeletal muscle and exerts a dominant toxic effect. Although there is evidence that TNPO3 is expressed in skeletal muscle (BioGPS portal for annotation resources (http://biogps.org) (Su et al., 2004), the role of TNPO3 in muscle is currently unknown. TNPO3 was originally identified as TNP-SR2, which encodes a nuclear membrane protein belonging to the importin beta family and transports serine/arginine (SR) rich proteins into the nucleus (Lai et al., 2000, 2001). TNPO3 was subsequently identified by genome-wide RNA interference knockdown as a HIV-dependency factor required for HIV1 infection at a stage between reverse transcription and integration of HIV in human cells (Brass et al., 2008; Konig et al., 2008). The protein mediates nuclear Downloaded from http://brain.oxfordjournals.org/ at Universidad de Valencia on May 13, 2013 Figure 3 Anti-TNPO3 and DAPI staining of muscle from affected individuals and controls. Immunofluorescence-stained muscle from two 64 March 29th, 2013 1516 | Brain 2013: 136; 1508–1517 cases with a congenital myopathy phenotype and a variable course, which can evolve into a severe and rapid progressive phenotype. Interestingly, these severely affected patients also manifested early joint and axial contractures similar to EmeryDreifuss syndrome, which raises the possibility of pathogenic mechanisms distinct from those involved in the typical cases. In addition, we have also observed very benign patients without complaints of weakness, but rather atypical features such as myalgia, exercise intolerance, and fatigue, mimicking a metabolic myopathy. This broad scenario of clinical nuances highlights the need to deepen the clinical evaluation of this extensive pedigree and other potential families with similar gene defects. In summary, in this report, we have provided an extensive update of the clinical and morphological features of LGMD1F and have identified a microdeletion mutation in the TNPO3 gene as a cause of this disorder. This finding expands our knowledge on the genetic bases of muscular dystrophies and suggests that other proteins of the nuclear envelope compartment may play a primary role in the pathogeneses of muscular dystrophies and other skeletal muscle-related disorders. Acknowledgements The authors thank the members of the family studied in this work for their collaboration, and Gisela Nogales-Gadea for scientific assistance. Funding This work was supported by the Spanish Instituto de Salud Carlos III [PS09/01591 to R.M., PI10/02628 to C.N., PI11/0842 to S.O., PI10/01970 to J.G., RD09/0076/00011 to the activities of Neurological Tissue Biobank, BIOBANCO del CHUVI]; the International Rare Diseases Research Consortium [SpainRDR]; the Conselleria de Economia e Industria, Xunta de Galicia [contract Isidro Parga Pondal to S.O.]; the U.S. National Institutes of Health (NIH) [R01 AR47989 to M.H.]. The CNAG thanks for core funding from the Spanish Ministerio de Economia y Competitividad and the Generalitat de Catalunya - Departament de Salut and Departament d’Economia i Coneixement. M.H. and A.K. also acknowledge support from NIH grants R01 HD057543 and R01 HD056103 from NICHD and the Office of Dietary Supplements (ODS), as well as U54 NS078059 from NINDS and NICHD, and from the Muscular Dystrophy Association USA. Downloaded from http://brain.oxfordjournals.org/ at Universidad de Valencia on May 13, 2013 import of the HIV pre-integration complex by binding the viral integrase, both in dividing and non-dividing cells (Christ et al., 2008). The C-terminus domain (CTD) of TNPO3 appears to be required for interactions with HIV1 integrase (Larue et al., 2012); therefore, the abnormal extension of the CTD domain is likely to interfere with its transport function. Because specific combinations of SR proteins are required for messenger RNA splicing and post-transcriptional processing (Bjork et al., 2009), the TNPO3 mutation may alter muscle transcripts raising the possibility that LGMD1F is RNA-mediated myopathy similar to, but mechanistically distinct from, myotonic dystrophy (Wheeler and Thornton, 2007; Tang et al., 2012). In addition, because mutations in the nuclear envelope proteins emerin and lamin A/C, are known to cause Emery-Dreifuss muscular dystrophy, the TNPO3 mutation causing LGMD1F extends the genetic spectrum of nuclear envelope-related myopathies. In support of this notion is the observation of filamentous inclusions and rimmed vacuoles in all three diseases (Fidzianska et al., 2004). We have noted filaments of 18 to 20 nm diameter both in nuclei and within the cytosol of myofibres of affected individuals. Filaments of similar thickness have been observed in several muscle disorders and are ascribed to accumulations of proteins, such as beta-amyloid, tau protein and ubiquitin (Askanas and Engel, 2006; Askanas et al., 2009). Myonuclear breakdown would entail the fragmentation of the nuclear membrane and contribute to the formation of pseudomyelin figures and membranous whorls, which correspond to the rimmed vacuoles seen by light microscopy. Interestingly, autophagic vacuoles, which we have observed in our affected subjects’ muscle, have been also noted in LGMD1D, which is due to mutations in DNAJB6. In that disease, the presence of autophagy is due to abnormal protein accumulation, which confers a dominant toxic function to the autophagic complex that contains the mutated co-chaperone (Sarparanta et al., 2012). Accordingly, autophagy may be contributing to LGMD1F. Clinically, LGMD1F was originally described as slowly progressive proximal symmetric weakness with predominantly lower limb onset, normal to mildly raised creatine kinase activity and myopathic electromyography features (Gamez et al., 2001). Great variability in age at onset, distribution of muscle involvement and severity was observed. Two clinical forms were delineated: a benign adult-onset form presenting in the third decade or later, and a juvenile form, beginning before age 15 years and leading to severe functional disability. Relative to the original report, the present study of a cohort of 30 patients with LGMD1F provides a longer and more systematic follow-up, as well as new information about affected individuals from younger generations. While confirming the core phenotype of LGMD1F, which is characterized by pelvic-femoral weakness and less severe and variable shoulder involvement, the new clinical data also demonstrate a broad clinical spectrum and novel clinical features of the disease. The disorder usually begins in childhood or adolescence, and less frequently in adulthood; and typically runs a benign course compatible with a normal working life. We noted mild hand and lower leg weakness producing a stereotyped posture while walking or standing on heels in virtually all patients. In addition, we observed less common but well-characterized presentations, including infantile-onset M. J. Melià et al. Supplementary material Supplementary material is available at Brain online. References Askanas V, Engel WK. Inclusion-body myositis: a myodegenerative conformational disorder associated with Abeta, protein misfolding, and proteasome inhibition. 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Brain 2013: 136; 1508–1517 66 February 12th, 2013 P07 Limb-Girdle Muscular Dystrophy and Inherited Myopathy Limb Girdle Muscular Dystrophy 1F: Clinical, Molecular and Ultrastructural Study (P07.032) Corrado Angelini1, Enrico Peterle2, Marina Fanin3, Giovanna Cenacchi4 and Vincenzo Nigro5 1 Neurosciences University of Padova Padova PD Italy 2 Neurosciences University of Padova Padova PD Italy 3 Neurosciences University of Padova Padova PD Italy 4 Pathology University of Bologna Bologna BO Italy 5 General Pathology University of Naples Naples NA Italy OBJECTIVE: To present clinical, muscle imaging, muscle histopathology, ultrastructural and genetic features in a large Italian-Spanish family with LGMD1F. BACKGROUND: The LGMDs are a heterogeneous group of hereditary disorders with weakness in proximal limb and/or distal muscles. To date 8 autosomal dominant forms of LGMD are known. LGMD1F clinical phenotype is characterized by a great variability, ranging from early onset, with a severe and rapidly progression to milder slow late-onset forms. The clinical and morphological features of patients with LGMD1F had not yet sufficiently characterized to suggest a specific etiology. DESIGN/METHODS: We collected the clinical history in 19/60 patients and expanded the family pedigree. Muscle biopsy histopathology, immunohistochemistry (desmin, myotilin, p62) and electron microscopy was investigated in one pair of affected patients (mother 1 biopsy, index patient 2 consecutive biopsies at 9 and 22 years). DNA from 4 patients was studied by Agilent MotorChip CGH array platform to identify the causative gene. RESULTS: Age of onset ranged from 2 to 35 years; in half cases there was hypotrophy both in proximal upper and in lower extremities in calf muscles. We noticed a discrepancy between the clinical severity and muscle biopsy involvement: the daughter (index case) has a more severe clinical course and increased muscle fiber atrophy whereas the mother has a compromised muscle histopathology (more muscle fiber variation, and autophagic changes). Accumulation of desmin and myotilin and p62-positive aggregates was observed. Electron microscopy revealed accumulation of myofibrillar bodies in muscle fibers. Muscle MRI in the index patient showed selective and severe atrophy in the vastus lateralis. CONCLUSIONS: Our morphological and ultrastructural data seem to suggest a myopathy phenotype similar to those described for Z-disk diseases. Although the specific genetic defect is still unknown, it is possible to hypothesize that LGMD1F might lead to disarrangement of desminassociated cytoskeletal network. Supported by: Telethon Italy, AFM (Association Francaise contre les Myopathies). Disclosure: Dr. Angelini has received personal compensation for activities with Genzyme as a member of the Advisory Board. Dr. Peterle has nothing to disclose. Dr. Fanin has nothing to disclose. Dr. Cenacchi has nothing to disclose. Dr. Nigro has nothing to disclose. 12 febbraio 2013 67 bs_bs_banner December 21st, 2012 Neuropathology 2012; ••, ••–•• doi:10.1111/neup.12003 Original Article Ultrastructural changes in LGMD1F Giovanna Cenacchi,1 Enrico Peterle,2 Marina Fanin,2 Valentina Papa,1 Roberta Salaroli1 and Corrado Angelini2,3 Department of Biomedical and Neuromotor Sciences, “Alma Mater” University of Bologna, Bologna, 2Department of Neurosciences, University of Padova, Padova and 3IRCCS S.Camillo, Venice, Italy 1 A large Italo-Spanish kindred with autosomal-dominant inheritance has been reported with proximal limb and axial muscle weakness. Clinical, histological and genetic features have been described. A limb girdle muscular dystrophy 1F (LGMD1F) disease locus at chromosome 7q32.1–32.2 has been previously identified. We report a muscle pathological study of two patients (mother and daughter) from this family. Muscle morphologic findings showed increased fiber size variability, fiber atrophy, and acid-phosphatasepositive vacuoles. Immunofluorescence against desmin, myotilin, p62 and LC3 showed accumulation of myofibrils, ubiquitin binding protein aggregates and autophagosomes. The ultrastructural study confirmed autophagosomal vacuoles. Many alterations of myofibrillar component were detected, such as prominent disarray, rod-like structures with granular aspect, and occasionally, cytoplasmic bodies. Our ultrastructural data and muscle pathological features are peculiar to LGMD1F and support the hypothesis that the genetic defect leads to a myopathy phenotype associated with disarrangement of the cytoskeletal network. Key words: electron microscopy, histopathology, limb girdle muscular dystrophy 1F, myofibrillar, myopathy. INTRODUCTION The limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of hereditary neuromuscular disorders with predominant or selective weakness in the proximal limb and/or distal muscles, having an estimated incidence of 1:100 000.1–3 The clinical phenotype is characterized by a great variability, ranging from early onset, with a severe and rapidly progressive clinical course, to milder forms, with a later onset and a slower progression. Autosomal- Correspondence: Giovanna Cenacchi, MD, Department of Biomedical and Neuromotor Sciences, Via Massarenti, 9, 40138 Bologna, Italy. Email: [email protected] Received 10 July 2012; revised and accepted 8 November 2012. dominant (AD) families representing less than 10% of the whole group of LGMDs,1–3 and to date, eight AD forms of LGMD, have been described.Among the AD forms, a large Italo-Spanish kindred with LGMD1F has been described with proximal limb and axial muscle weakness.4,5 Clinical, histological and genetic features have been described in 5/32 patients. In this family, the disease locus has been mapped to chromosome 7q32.1–32.2, but no mutation was detected in filamin C, a possible candidate gene in this chromosomal region, which encodes for actin binding protein highly expressed in muscle.5 The clinical and morphological features of two patients with LGMD1F are here described since the disease was not yet sufficiently characterized to suggest a specific etiologic category. We report a muscle pathological study of two patients (mother and daughter) from this Italo-Spanish family. An ultrastructural and immunofluorescence approach has been performed to investigate the pathogenetic mechanism. MATERIALS AND METHODS Muscle biopsy histopathology was investigated in one pair of affected patients (mother, Case 1; daughter, Case 2). Childhood onset was observed in Case 2 (6–7 years) with a faster weakness progression; at 22–23 years the patient showed difficulty in rising with mild respiratory and swallowing impairment. In comparison, in Case 1 clinical symptoms were relatively mild until the age of 32 years; then the progression rate was slow. Skeletal muscle biopsies were performed in vastus lateralis after obtain patient consent. Muscle specimens were oriented, snap-frozen in liquid nitrogen-chilled isopentane and the cryostat-cut sections were stained using a panel of routine histochemical methods: HE, modified Gomori trichrome, reduced nicotinamide-adenine-dinucleotide-tetrazolium-reductase (NADH-TR), combined cytochrome oxidase (COX) and succinic dehydrogenase (SDH), adenosine triphosphatases (ATPases) and acid phosphatase. Immunofluorescence analysis for desmin (MAB1698 Chemicon, Temecula, CA, © 2012 Japanese Society of Neuropathology 68 December 21st, 2012 2 G Cenacchi et al. US; dilution 1:50) and myotilin (RSO34, Novocastra, Newcastle, UK; dilution 1:50) and double-labeling for LC3 (2775,Cell SignalingTechnology,Danvers,MA,US;dilution 1:100) and p62 (Gp62C, Progen Biotechnik, Heidelberg, Germany; dilution 1:200) were performed using immunofluorescence microscopy. Fresh tissues from each biopsy were fixed in 2.5% glutaraldehyde in cacodylate buffer, post-fixed in 1% OsO4 in the same buffer, dehydrated in graded ethanol, and embedded in araldite. Semithin sections were stained with toluidine blue.Thin sections, stained with uranyl acetate and lead citrate, were examined with a Philips 400T transmission electron microscope. A B C D RESULTS Morphologic findings by HE showed increased fiber size variability; the fibre atrophy was more prominent in Case 2, whereas endo- and perimisial connective tissue, and acidphosphatase-positive areas were more pronounced in Case 1 (Fig. 1). Immunofluorescence for desmin and myotilin showed accumulation of reaction in myofibrillar structures in some fibers of both patients (Fig. 2). Double-labelling for p62 and LC3 demonstrated increased protein aggregates (p62-positive) within some atrophic fibers (Fig. 3); LC3 labelling, used as a marker of autophagosomes, was Fig. 1 Muscle biopsy sections stained for HE (A,B), and acid phosphatase (C,D) of Case 2 (A,C) and Case 1 (B,D). Note fiber size variability, diffuse fiber atrophy in Case 2 (A, B) and accumulations of acid phosphatase-positive material (C,D). Microscope magnification ¥200. Fig. 2 Muscle biopsy sections from Case 2 (A,C,D) and Case 1 (B) immunostained for desmin (A) or myotilin (B–D). Note accumulation of cytoskeletal (desmin) or sarcomeric (myotilin) proteins, occupying a relatively large cytoplasmic area of isolated myofibers. Microscope magnification ¥200. © 2012 Japanese Society of Neuropathology 69 December 21st, 2012 3 Ultrastructure of LGMD1F Fig. 3 Double immunofluorescence analysis on muscle biopsy sections using antibodies against p62 (green) and LC3 (red) (counterstain of nuclei with 4′,6-diamino-2phenylindole, blue) in Case 2 (A,B) and Case 1 (C,D). Note accumulation of p62-positive protein aggregates in some atrophic muscle fibers in both patients. Fig. 4 Ultrastructural analysis showing atrophic fiber characterized by many polymorphic mitochondria with paracrystallinelike inclusions (arrow) (A) and myelinoid body adjacent to the subsarcolemmal nucleus (B) in Case 2. Myofibrillar alterations lead to architectural disarray of muscle fibers in Case 1 (C), and to accumulation of electrondense material of possible Z-linederivation in the subsarcolemmal areas in Case 2 (D). At higher magnification, the electron-dense material appears as a granulo-filamentous pattern (d-inset). Scale bar = (A) 2000 nm; (B) 1000 nm; (C) 2000 nm; (D) 5000 nm (inset: 2000 nm). A B C D mild and diffuse (Fig. 3). The ultrastructural study confirmed fiber atrophy (Fig. 4), abnormal mitochondria accumulations with only rare paracrystalline-like inclusions (Fig. 4) and autophagosomal vacuoles containing cytoplasmic debris and myelinoid bodies (Fig. 4). No tubulofilamentous cytoplasmic or nuclear inclusions were detected. Many alterations of myofibrillar component were easily detected, such as prominent disarray (Fig. 4), rodlike structures with granular aspect (Fig. 4), and occasional filamentous cytoplasmic bodies. No ultrastructural differences were appreciated between the two cases. DISCUSSION The histopathological data and the electron microscopic findings from our patients extend previous results described for this LGMD1F family.4,5 In the original report, the morphologic findings were abnormal fiber size with degenerative aspect and prominent rimmed vacuoles.4 COX–SDH stain showed about 5–30% of COX-negative fibres. The ultrastructural description has been performed in three cases.4 That study focused on the presence of both autophagosomes with cytoplasmic bundles of filaments in © 2012 Japanese Society of Neuropathology 70 December 21st, 2012 4 one case, and a large amount of degenerating mitochondria, which were considered a secondary unspecific feature.4 Changes related to mitochondria and autophagosomal vacuoles are commonly seen in a wide variety of myopathies such as inclusion body myositis (IBM)6,7 and oculopharyngeal nuscular dystrophy (OPMD).8–10 Whereas abnormalities of p62 and ubiquitin-binding proteins might be signals of protein degradation, in our cases protein aggregates were associated with p62 and diffuse LC3. Abnormalities of NBR1 (neighbor of BRCA1 gene 1), a novel autophagyassociated protein, could be a useful tool to further study chronic progressive myopathies.7 We did not identify in our patients’ biopsies, neither nuclear nor cytoplasmic tubulofilament inclusions, which are considered specific for IBM if associated with the presence of rimmed vacuoles.6,7 We did not see intranuclear tubulofilaments arranged in tangles or palisades which are described in the OPMD.9,10 The present ultrastructural observations highlight in both cases severe modifications of myofibrillar filaments which appear disorganized with granular rod-like structures, cytoplasmic bodies and myofibrillar disarray with focal perpendicular arrangement. The presence of myofibrillar disarray and granulo-filamentous material is likely to derive from the Z-line, and this is supported by immunofluorescence for desmin and myotilin, which are usually observed in myofibrillar myopathies. Among myopathies characterized by myofibrillar derangement, the myofibrillar myopathies have been well described.11–14 They are a group of muscle disorders associated with similar morphologic features consisting of myofibrillar disorganization originating from the Z-disk followed by accumulation of myofibrillar degradation products. They may be defined by the presence of rimmed vacuoles, associated with ectopic expression of multiple proteins that include desmin, neural cell adhesion molecule, plectin, gelsolin, ubiquitin, Xin, TAR DNA-binding protein 43 and cochaperones, including aB-crystallin, heat shock protein-27.11–14 Myotilinopathy (LGMD1A) and filaminopathy have been reported as a subset of myofibrillar myopathy. Both myotilinopathy and filaminopathy (the so-called Z-disk diseases) exhibit the morphological findings typical of myofibrillar myopathy with filament accumulation including Z-disk alterations.12,13 Particularly in filaminopathy, strongly positive for filamin C, ultrastructural examination revealed major myofibrillar abnormalities, including accumulation of desmin-positive granulo-filamentous material. In addition, also large autophagic vacuoles and mitochondrial aggregates in the abnormal fiber regions were observed.15–17 The disease mechanism in filaminopathy is still unclear, but it may involve structural alterations of the Z-disk caused by dysfunctional proteins or their abnormal accumulation due to defective degradation.12,13 In particular, among cytoskeletal proteins, desmin, with its binding partners, forms a three- G Cenacchi et al. dimensional scaffold around Z-disks, thereby interlinking with myofibrils and nuclei, mitochondria and sarcolemma. Several studies demonstrated that the filamentous desmin network plays an essential role in the subcellular positioning and function of mitochondria.11–14 Indeed,mitochondrial accumulation has been clearly showed in LGMD1F, and it was confirmed also in the present two cases, where rare paracrystalline inclusions were found similar to those previously described.4 The contemporary presence of autophagosomes and several myelin figures is the morphological substrate of a protein quality-control disturbance related to the ubiquitin–proteasome system (UPS) and the autophagiclysosomal pathway.11 A recent study showed that desmin mutants impair the proteolytic function of the UPS and the autophagic–lysosomal pathway (types 1 and 2 of programmed cell death).11 Electron microscopy is useful in the diagnostic workup of chronic myopathies identifying pathological protein aggregation, cytoplasmic/spheroid bodies, and signs of myofibrillar degeneration, such as sarcoplasmic granulofilamentous material, autophagic vacuoles and myelin-like whorls.11 To address the alteration of the Z-line, both degeneration, streaming, irregularities and Z-line loss should be detected. Furthermore, additional features can be found, such as depletion or accumulation of mitochondria. Our morphological and ultrastructural data seem to suggest in our LGMD1F cases a myopathy phenotype similar to those described for Z-disk diseases.Although the genetic defect is still under investigation, it is possible to hypothesize that the mutant protein in LGMD1F might lead to disarrangement of desmin-associated cytoskeletal networks. CONFLICT OF INTEREST The authors declare that they have no conflict of interest. REFERENCES 1. Broglio L, Tentorio M, Cotelli MS et al. Limb-girdle muscular dystrophy-associated protein diseases. Neurologist 2010; 16: 340–352. 2. Guglieri M, Straub V, Bushby K, Lochmuller H. Limbgirdle muscular dystrophies. Curr Opin Neurol 2008; 21: 576–584. 3. Nigro V, Aurino S, Piluso G. Limb girdle muscular dystrophies: update on genetic diagnosis and therapeutic approaches. Curr Opin Neurol 2011; 24: 429–436. 4. Gamez J, Navarro C, Andreu AL et al. Autosomal dominant limb-girdle muscular dystrophy. A large kindred with evidence for anticipation. Neurology 2001; 56: 450–454. © 2012 Japanese Society of Neuropathology 71 December 21st, 2012 5 Ultrastructure of LGMD1F 5. Palenzuela L, Andreu AL, Gàmez J et al. A novel autosomal dominant limb-girdle muscular dystrophy (LGMD1F) maps to 7q32.1-32.2. Neurology 2003; 61: 404–406. 6. Askanas V, Engel WK. Sporadic inclusion-body myositis: conformational multifactorial ageing-related degenerative muscle disease associated with proteasomal and lysosomal inhibition, endoplasmic reticulum stress, and accumulation of amyloid-b42 oligomers and phosphorylated tau. Presse Med 2011; 40: 219–235. 7. D’Agostino C, Nogalska A, Cacciottolo M, Engel WK, Askanas V. Abnormalities of NBR1, a novel autophagy-associated protein, in muscle fibers of sporadic inclusion-body myositis. Acta Neuropathol 2011; 122: 627–636. 8. Gambelli S, Malandrini A, Ginanneschi F et al. Mitochondrial abnormalities in genetically assessed oculopharyngeal muscular dystrophy. Eur Neurol 2004; 51: 144–147. 9. Van Der Sluijs BM, Hoefsloot LH, Padberg GW, Van Der Maarel SM, Van Engelen BG. Oculopharyngeal muscular dystrophy with limb girdle weakness as major complaint. J Neurol 2003; 250: 1307–1129. 10. Schröder JM, Klossok T, Weis J. Oculopharyngeal muscle dystrophy: fine structure and mRNA 11. 12. 13. 14. 15. 16. 17. expression levels of PABPN1. Clin Neuropathol 2011; 30: 94–103. Schröder R, Schoser B. Myofibrillar myopathies: a clinical and myopathological guide. Brain Pathol 2009; 19: 483–492. Selcen D. Myofibrillar myopathies. Curr Opin Neurol 2010; 23: 477–481. Selcen D. Myofibrillar myopathies. Neuromuscul Disord 2011; 21: 161–171. Montse O, Odgerel Z, Martınez A et al. Clinical and myopathological evaluation of early- and late-onset subtypes of myofibrillar myopathy. Neuromuscul Disord 2011; 21: 533–542. Vorgerd M, van der Ven PFM, Bruchertseifer V et al. A Mutation in the dimerization domain of Filamin C causes a novel type of autosomal dominant myofibrillar myopathy. Am J Hum Genet 2005; 77: 297–304. Kley RA, Hellenbroich Y, van der Ven PFM et al. Clinical and morphological phenotype of the filamin myopathy: a study of 31 German patients. Brain 2007; 130: 3250–3264. Shatunov A, Olive M, Odgerel Z et al. In-frame deletion in the seventh immunoglobulin-like repeat of filamin C in a family with myofibrillar Myopathy. Eur J Hum Genet 2009; 17: 656–663. © 2012 Japanese Society of Neuropathology 72 August 30th, 2012 806 Abstracts / Neuromuscular Disorders 22 (2012) 804–908 Department of Genetics, Sydney, Australia; 5 Alfred Hospital, Department of Anatomical Pathology, Melbourne, Australia; 6 University of Western Australia, Centre for Medical Research, Perth, Australia; 7 University of Western Australia, Centre for Neuromuscular and Neurological Disorders, Perth, Australia The dystrophinopathies are allelic muscular dystrophies caused by Xlinked recessive mutations in dystrophin, with only rare reports of asymptomatic adult males. The static cognitive impairment seen in dystrophinopathies is thought to be due to altered expression of dystrophin isoforms, but has only once been described in the absence of muscle weakness. We identified a cohort of patients with unexpected copy number variants (CNV) in the dystrophin gene, on microarrays performed for developmental delay or intellectual disability, in whom muscle weakness was minimal or absent. Subjects with a dystrophin CNV referred to the neurology or genetics departments at RCH Melbourne or GHSV were assessed. An additional family was identified from the CHW, and included. Twelve probands had a CNV in the dystrophin gene on micro-array testing. Eight (seven male, one female; seven deletions, one duplica-tion), age 0–9 years, had atypical phenotypes as described above. CNVs were found in 10 family members (five males and five females), including three asymptomatic adult males. In all but one family, MLPA confirmed loss of exons. Muscle weakness was absent or minimal. Serum CK was normal or mildly elevated. Muscle biopsy revealed morphologically nor-mal muscle with normal dystrophin immunoreactivity. Microarray testing has revealed an extended spectrum of clinical phenotypes associated with mutations in dystrophin, that may include isolated developmental delay and asymptomatic individuals. Further study is required to understand the molecular basis of the apparent absence of muscle pathology in these patients, and the relationship of the dystrophin deletion to cognitive impairment. http://dx.doi:10.1016/j.nmd.2012.06.016 D.O.3 Next generation sequencing applications are ready for genetic diagnosis of muscular dystrophies M. Savarese 1, A. Torella 1, M. Mutarelli 2, M. Dionisi 2, T. Giugliano 3, G. Di Fruscio 3, M. Iacomino 3, A. Garofalo 3, S. Aurino 3, F. Del Vecchio Blanco 3, G. Piluso 3, L. Politano 4, M. Fanin 5, C. Angelini 5, V. Nigro 3 1 Seconda Università degli Studi di Napoli, Laboratorio di Genetica Medica, Dipartimento di Patologia Generale, Napoli, Italy; 2 TIGEM, Telethon Institute of Genetic and Medicine, Napoli, Italy; 3 Seconda Università degli Studi di Napoli, Dipartimento di Patologia Generale, Napoli, Italy; 4 Seconda Università degli Studi di Napoli, Cardiomiologia e Genetica Medica, Napoli, Italy; 5 Università degli Studi di Padova, Department of Neurosciences, Padova, Italy Next generation sequencing (NGS) is having a tremendous impact on our knowledge of different aspects of biology. It can be also very powerful to study patients with heterogeneous genetic conditions, like muscular dystrophies. First, to identify new genes using “exome resequencing”. Second, to diagnose mutations in all the known causative genes, when used as targeted approach. Third to obtain a knowledge of the impact of mutations on mRNA expression and splicing in diseased muscle. We used NGS to identify new genes by whole exome sequencing. We sequenced the whole exome of four family members with LGMD1F separated by up to eleven meioses and identified a single shared novel heterozygous frame-shift variant. This causes a nonstop change in the Transportin 3 (TNPO3) gene that encodes a member of the importin-b super-family. To reach the second task, we first recruited 160 familial cases of nonspecific limb-girdle muscular dystrophies with apparent autosomal inheritance. All DNA samples were first enriched for 486,480 bp, covering 2447 exons of 98 genes by using the Haloplex tech- nology with the use of barcodes. We the performed pooled NGS of all samples and identified a number of mutations, then verified by Sanger sequencing. Cases were also studied by the Agilent MotorChip CGH array version 3.0 to identify deletions or duplications. Finally, in selected cases, we performed the RNA-Seq starting from a muscle biopsy sample. We converted mRNA to cDNA and purified it by a customized SureSelect Target Enrichment System, focused on the same 98 mRNAs. The probes had a 4 coverage with a total target of 1.41 Mb of sequences/ sample. These cDNAs were sequenced using barcodes trying to obtain an average sequencing coverage of at least 100. Our results confirm that there is a very high genetic heterogeneity in muscular dystrophies and that NGS-based DNA and RNA testing are ready for diagnostic use. http://dx.doi:10.1016/j.nmd.2012.06.017 D.O.4 Next generation sequencing for genetic diagnosis and gene identification in myopathies J. Bohm 1, N. Vasli 1, U. Schaffer 1, S. Le Gras 2, B. Jost 2, N.B. Romero 3, N. Levy 4, E. Malfatti 3, V. Biancalana 1, J. Laporte 1 1 IGBMC, Translational Medecine, Illkirch, France; 2 IGBMC, Illkirch, France; 3 Insitut de Myologie, Unite de Morphologie Neuromusculaire, Paris, France; 4 Faculté de Médecine de Marseille, Inserm UMRS 910, Marseille, France Myopathies are rare diseases with a high impact on patients, fami-lies and the health care system. Despite tremendous efforts, about half of patients do not have a molecular diagnosis. This is mainly due to genetic heterogeneity, the fact that very large genes known to be mutated in myopathies are difficult to screen, and the presence of yet unidentified genes. We provide the proof-of-principle that next genera-tion sequencing (NGS) can be used for molecular diagnosis, to screen large genes, and to identify novel genes. For molecular diagnosis, we used a custom capture library to enrich the coding sequence and intron–exon boundaries of 267 genes known to be mutated in neuro-muscular diseases. We could detect all known mutations in previously characterized patients, including homozygous, heterozygous, exonic, intronic, point, small indel mutations and a large deletion. The cost to sequence these 267 genes is lower than to test one gene by the con-ventional Sanger method. We then tested several patients without molecular diagnosis and could find mutations in several of them includ-ing mutations in TTN, the largest human gene. We also used exome sequencing in different myopathy cohorts and identified diseasecausing mutations in RYR1 and NEB genes, large genes not screened on rou-tine if RNA is not available. Phenotypes of patients with RYR1 mutations were very heterogeneous, supporting that NGS broadens genotype– phenotype correlations and represents an unbiased approach to investigate mutation/gene frequency in myopathies. Next we used exome and genome sequencing to identify disease-causing genes in spe-cific myopathies for which no causative genes were previously known. We found mutations either in genes previously linked to other myopa-thies or in novel genes. Examples will be presented. Next generation sequencing will accelerate mutation discovery for the benefit of patient diagnosis and a better understanding of muscle function under normal and pathological conditions. http://dx.doi:10.1016/j.nmd.2012.06.018 D.O.5 A combination of linkage analysis and exome sequencing identifies a new gene for X-linked Charcot–Marie–Tooth neuropathy 73 June 12th, 2012 Muscle disorders Tuesday, June 12, 2012, 11:30 - 12:30 New quantitative MRI indexes useful to investigate muscle diseases C. Angelini, M. Fanin, E. Peterle (Padova, IT) Objectives: We propose new types of quantitative measurement to evaluate muscle atrophy: the quadriceps index (QI) and the left vastus lateralis index (VLI), measuring by MRI their area. Methods. We have used T1 sequences on thigh muscle MRI, at about 15 cm from the head of the femur (second slide of MRI in lower extremities). In these sequences we measured the muscle area of the left quadriceps femoris and of the left vastus lateralis. These measurement were carried out in 11 patients with various types of myopathies i.e. two cases of lipid storage myopathies, 1 amyotrophic lateral sclerosis, 1 facio-scapulo-humeral dystrophy, 1 myofibrillar myopathy, 1 metabolic myopathy, 2 patients with LGMD2A, 1 patient with LGMD1F, 1 localized myositis ossificans, 1 aspecific myopathy. Muscle biopsies of these patients were further investigated by morphometry and molecular markers of atrophy or autophagy i.e.MURF, LC3. Results: We performed the measurement of muscle area of quadriceps femoris (Q.I) in 11 patients, that resulted in average 3711 mm2 ± SD 792. In this group of patients we have identified two subgroups, one including 5 patients with a high degree of muscle atrophy (highly atrophic group), whose values ranged from 2400 to 3400 mm2 (mean 2966), and one including 6 patients with a low degree of atrophy (low atrophic group), whose values ranged from 3700 to 5000 mm2 (mean 4332). The measurement of muscle area of vastus lateralis in 11 patients was in average 963 mm2 ± 303. In the atrophic subgroup the values ranged from 400 to 900 mm2 (mean 658.7), while in the normal sub-group the values ranged from 900 to 1400 mm2 (mean 1217.8). Conclusion: Both the quadriceps and the vastus lateralis indexes appear useful to evaluate muscle atrophy in LGMDs, ALS and metabolic myopathies: a high degree of atrophy of QI was found in calpainopathy, motor neuron disease and Limb Girdle Muscular Dystrophy type 1F, the measurement of the VLM appeared less specific since it includes a larger area. Both these quantitative indexes obtained by muscle MRI, could be used as clinical outcomes of treatment in neuromuscular disorders in order to follow up and study natural history or the effect of various type of treatments (steroids, carnitine, etc.). A promising field of investigation appears the correlation of imaging indexes with other atrophy parameters obtained in muscle biopsy, i.e with the cross sectional area or fibers or with molecular markers of atrophy and autophagy. 74 2012 P115 IDENTIFICAZIONE DI NUOVI GENI COINVOLTI NELLE DISTROFIE MUSCOLARI DEI CINGOLI MEDIANTE ARRAYS E SEQUENZIAMENTO DI NUOVA GENERAZIONE (NGS) 1 3 2 3 3 2 1 A. Torella , F. Del Vecchio Blanco , M. Dionisi , A. Garofalo , M. Iacomino , M. Mutarelli , M. Savarese , G. Piluso 1 1 , V. Nigro Dip. di Patologia Generale-Lab. di Genetica Medica, Seconda Università degli Studi di Napoli, Telethon Institute of Genetics and Medicine (TIGEM) 2 Telethon Institute of Genetics and Medicine (TIGEM) 3 Dip. di Patologia Generale-Lab. di Genetica Medica, Seconda Università degli Studi di Napoli 1 Campioni di DNA di soggetti affetti da distrofia muscolare sono stati da noi analizzati per i geni responsabili di LGMD: il 30% dei pazienti presentava una mutazione nel gene CAPN3, il 10% nel gene DYSF , il 10% nei geni dei sarcoglicani e un altro 10% negli altri geni noti LGMD:LGMD2A-N. Una significativa percentuale di pazienti con LGMD non aveva alcuna mutazione nei 18 geni LGMD scoperti finora. In particolare, il 40% dei pazienti non ha una diagnosi molecolare. La spiegazione è da ricercare nell'elevata eterogeneita' genetica. Le tecniche tradizionali presentano l'inconveniente di concentrare la ricerca delle mutazioni su un singolo gene alla volta. Inoltre, gli attuali esami genetici sono lunghi, costosi e senza effetti. I nuovi potenti approcci per lʼanalisi del DNA, come la next-generation sequencing (NGS) sono in procinto di rivoluzionare il campo con un singolo strumento in grado di analizzare lʼintero genoma umano per molte volte. La nostra ricerca ha combinato analisi di linkage basata su SNP array e la tecnologia NGS al fine di scoprire mutazioni “orfane” di LGMD. I pazienti oggetto di studio sono stati selezionati secondo i seguenti criteri: a)diagnosi clinica di LGMD; b)diagnosi molecolari non concluse, c)la maggior severità della malattia; Tutti gli altri casi sono stati studiati mediante 8x60k Motor Chip, un array-CGH basato su oligonucleotidi con una copertura esonica completa dei geni coinvolti nelle malattie neuromuscolari che permette di individuare delezioni o duplicazioni deleterie. Gli esomi di 16 soggetti appartenenti a 7 diverse famiglie sono stati sequenziati mediante NGS utilizzando la piattaforma SOLID e, in parallelo, (1 famiglia) lʼ Illumina HiSeq2000. Un certo numero di mutazioni sono state identificate. In particolare quattro membri affetti di una famiglia con ereditarietà AD (LGMD1F) presentano una singola mutazione (frame-shift) non trovata negli altri membri della famiglia o controlli. In una seconda famiglia LGMD con ereditarietà AR abbiamo recentemente identificato una mutazione missenso in omozigosi nel gene ACADVL che è condivisa da tutti i membri affetti della famiglia e da altri pazienti provenienti dalla stessa area geografica. 75 October 30th, 2011 Acta Myologica • 2011; XXX: p. 168 ADDENDUM Proceedings of the XI Congress of the Italian Association of Myology Cagliari, May 2011 LGMD 1(F) - A pathogenetic hypothesis based on histopathology and ultrastructure G. Cenacchi, E. Peterle, L. Tarantino, V. Papa, M. Fanin, C. Angelini Clinic Department of Radiologic and histopathologic Sciences, University of Bologna, Department of Neurosciences and VIMMM, University of Padua A large Spanish kindred with apparently autosomal dominant inheritance has been reported with proximal limb and axial muscle weakness. Clinical, histological and genetic features have been described in 5/32 patients. A novel LGMD disease locus at chromosome 7q32.1-32.2 has been identified, but any defects were detected in filamin C, a gene candidate from this chromosomal region encoding actin binding protein highly expressed in muscle. We report a clinico-pathological study of two patients (mother and daughter) from the same Spanish family. Age at onset was in the teens: earlier onset in the daughter with a faster weakness progress confirms an apparent genetic anticipation. Morphologic findings were similar in both cases: H&E notices increased fiber size variability, fiber atrophy, endo- and perimisial connective tissue, and acid-phosphatase positive vacuoles. The ultrastructural study confirmed fiber atrophy, abnormal mitochondria accumulations and autophagosomal vacuoles containing cell debris and pseudomyelin figures: no filamentous inclusions were detected which are usually associated with a HIBM. Many alterations of myofibrillar component were easily detected such as prominent disarray, rod-like structures with granular aspect, and occasionally cytoplasmic bodies. Our morphological data support the hypothesis that other actin-encoding proteins such as FSCN3, and KIAA0265 from the same critical region may represent attractive candidate genes in the LGMD 1(F) pathogenetic mechanism. Abstract omitted in Acta Myologica, Vol. XXX, June 2011 168 76 August 2003 77 August 2003 78 August 2003 79 August 8th, 2000 Autosomal dominant limb-girdle muscular dystrophy A large kindred with evidence for anticipation J. Gamez, MD; C. Navarro, MD; A.L. Andreu, MD; J.M. Fernandez, MD; L. Palenzuela, MS; S. Tejeira, MS; R. Fernandez–Hojas, MS; S. Schwartz, MD, PhD; C. Karadimas, PhD; S. DiMauro, MD; M. Hirano, MD; and C. Cervera, MD Article abstract—Background: Fourteen genetically distinct forms of limb-girdle muscular dystrophy (LGMD) have been identified, including five types of autosomal dominant LGMD (AD-LGMD). Objective: To describe clinical, histologic, and genetic features of a large Spanish kindred with LGMD and apparent autosomal dominant inheritance spanning five generations. Method: The authors examined 61 members of the family; muscle biopsies were performed on five patients. Linkage analysis assessed chromosomal loci associated with other forms of AD-LGMD. Results: A total of 32 individuals had weakness of the pelvic and shoulder girdles. Severity appeared to worsen in successive generations. Muscle biopsy findings were nonspecific and compatible with MD. Linkage analysis to chromosomes 5q31, 1q11-q21, 3p25, 6q23, and 7q demonstrated that this disease is not allelic to LGMD forms 1A, 1B, 1C, 1D, and 1E. Conclusions: This family has a genetically distinct form of AD-LGMD. The authors are currently performing a genome-wide scan to identify the disease locus. NEUROLOGY 2001;56:450–454 The limb-girdle muscular dystrophies (LGMD) comprise a genetically diverse group of muscle disorders with predominantly proximal limb and axial muscle weakness. Because of molecular genetic discoveries and improved clinical criteria, the classification and nomenclature of LGMD have evolved over the last decade.1,2 Many LGMD disorders are autosomal recessive traits, but at least five well-characterized forms have been reported in recent years.3-12 We studied a large Spanish kindred with 32 affected individuals and apparently autosomal dominant inheritance spanning five generations. Here, we describe the clinical phenotype and morphologic findings in five patients who underwent muscle biopsy, and preliminary genetic investigations of this new type of autosomal dominant LGMD (AD-LGMD). Patients and methods. A total of 61 individuals from five generations of a family from eastern Spain were examined. Serum creatine kinase (CK), aspartate aminotransferase (ASAT), and alanine aminotransferase (ALAT) determinations were performed on all subjects. Twelve underwent neurophysiologic examinations, and five had muscle biopsy. Other investigations included electrocardiography (ECG) in 12 patients, echocardiography in six patients, and MRI of the brain in two patients. Subjects were considered affected when clinical exami- nation revealed a characteristic pattern of muscular weakness, primarily affecting the pelvic and shoulder girdles. Muscle strength was assessed using the British Medical Research Council (MRC) Scale; 26 muscle groups were examined bilaterally. Functional ability was measured according to the scales designed by Vignos and Brooke.13-14 Age at onset was determined using a standardized clinical questionnaire form asking clinically affected individuals to identify their first symptoms from a list that included a waddling gait and difficulty in climbing stairs, raising hands above the head, lifting, running, and rising from a chair or squatting position. A total of 32 family members were clinically affected. In figure 1, individuals are identified by generation number (Roman numerals) followed by birth order position, reading from left to right (Arabic numerals). Skeletal muscle biopsy specimens from the deltoid or vastus lateralis were oriented, snap-frozen in liquid nitrogen-chilled isopentane and the cryostat-cut sections were stained using standard histochemical methods. Immunohistochemistry was performed using the following antibodies: desmin and vimentin (Biogenex, CA), ubiquitin (DAKO, DK), Tau protein and Beta-amyloid (Sigma, MO), and dystrophin, sarcoglycans, and the amino terminal of utrophin (DRP2, Novocastra Laboratories, Newcastle upon Tyne, UK). A small portion of each sample was fixed in glutaraldehyde and processed for ultrastructural examination. Genetic linkage studies were performed to exclude chro- From the Department of Neurology (Drs. Gamez and Cervera) and Centre d’ Investigacions en Bioquimica i Biologia Molecular (Drs. Andreu and Schwartz, and L. Palenzuela), Hospital Vall d’ Hebron, Barcelona; Department of Pathology and Neuropathology (Dr. Navarro, S. Tejeira, and R. Fernandez–Hojas) Hospital do Meixoeiro; Department of Clinical Neurophysiology (Dr. Fernandez), Hospital Xeral-Cies, Vigo, Spain; and H. Houston Merritt Clinical Research Center for Muscular Dystrophy and Related Diseases (Drs. Karadimas, DiMauro, and Hirano), Department of Neurology, Columbia University College of Physicians and Surgeons, New York. Supported by the Spanish Fondo de Investigación Sanitaria (FIS 00/797). Received June 8, 2000. Accepted in final form October 27, 2000. Address correspondence and reprint requests to Dr. Josep Gamez, Department of Neurology, Hospital Gral, Vall d’Hebron, Passeig Vall d’Hebron, 119-125, 08035 Barcelona, Spain; e-mail: [email protected] 450 Copyright © 2001 by AAN Enterprises, Inc. 80 August 8th, 2000 Figure 1. Drawing of the pedigree. Clinically affected members are shown in black. Roman numerals indicate generation number, and Arabic numerals birth order position within the generation. mosomal loci associated with other forms of AD-LGMD (LGMD1), hereditary inclusion body myopathy (HIBM), and Bethlem myopathy. Blood samples were taken from all 61 members of the pedigree after informed consent. After DNA extraction from the blood buffy coats, a set of fluorescent-labeled microsatellite simple sequence repeat (SSR) markers spanning the five known loci of LGMD1 were genotyped using an ABI Prism 310 Genetic Analyzer (Perkin Elmer, Foster City, CA). In addition, SSR markers encompassing the loci of autosomal recessive HIBM (AR-HIBM),15 autosomal dominant HIBM (AD-HIBM),16 Bethlem myopathy,17-18 and facioscapulohumeral dystrophy (FSHD)19 were genotyped. These SSR markers were as follows: 1. LGMD 1A (5q31): D5S410, D5S436, D5S2115, and D5S471 2. LGMD 1B (1q11-q21): D1S218, D1S196, D1S2878, D1S484, D1S2635, D1S498, D1S252, and D1S2726 3. LGMD 1C (3p25): D3S1277, D3S1266, D3S2338, D3S1263, D3S1304, and D3S1297 4. LGMD 1D (6q23): D6S308, D6S292, D6S262, and D6S287 5. LGMD 1E (7q): D7S427, D7S2465, D7S798, D7S636, and D7S661 6. AR-HIBM (9p1-q1): D9S161, D9S1817, D9S273, and D9S175 7. AD-HIBM (17p13.1): D17S938, D17S1852, D17S799, and D17S921 8. Bethlem myopathy (2q37 and 21q22.3); D2S206, D2S338, D2S125, D21S1252, D21S1255, and D21S266 9. FSHD (4q35): D4S2920, D4S1535, D4S2924, and D4S426 Two-point (marker-to-disease) analysis was performed with the MLINK option of FASTLINK 4.0.20 The disease was considered autosomal dominant with 90% penetrance. The disease gene frequency was estimated at 1/100,000. Marker allele frequencies were estimated by allele counting of all genotyped subjects. The LINKMAP option was used for three-point analyses. Marker order and intermarker distances were based on the Genethon linkage map. Results. Characteristic muscle weakness predominantly involving the pelvic and shoulder girdle proximal muscles was shown in 32 individuals (15 men, 17 women) between the ages of 7 and 66 years (mean 34.5, SD 14.4 years). Age at onset ranged from less than 1 to 58 years (mean 16.3, SD 15.5 years). Two groups were delineated based on age at onset: a juvenile form, with onset before age 15 (65.6% of patients) and an adult-onset form, starting around the third or fourth decade (28.0%). Symptoms of pelvic girdle muscular weakness were noted at onset in 81.1% of cases. Commonly affected muscles were the iliopsoas (96.0%), gluteal (75.0%), hip adductors (71.8%), deltoid (90.6%), biceps brachii (68.7%), paraspinal (65.6%), and neck flexors (62.5%). Pelvic girdle impairment was more severe and occurred earlier than in the shoulder girdle. Proximal muscle weakness ranged from MRC grade 0 to 4/5, with symmetric distribution. Distal weakness appeared late in the disease’s course or accompanied initial presentation in severely affected juvenile-onset patients, frequently affecting the extensor digitorum, tibialis anterior, and toe extensor muscles. Six patients had scapular winging. Two juvenile-onset patients showed mild facial weakness 10 years after onset. Early-onset patients had generalized muscular wasting, predominantly involving the quadriceps, gluteus, deltoid, biceps, infraspinatus, and supraspinatus muscles (figure 2). Early joint contractures were not present. Three individuals developed Achilles tendon contractures late in the disease’s course. Four subjects showed scoliosis or hyperlordosis. All belonged to the juvenile-onset group. Respiratory muscles were clinically affected in four patients with juvenile-onset form. Mean forced vital capacity in these patients was 38.6% of predicted values. No patient had ptosis, ophthalmoparesis, dysphagia, speech disturbances, calf hypertrophy, myalgia, or intellectual deterioration. Weakness progress during the 8-year follow-up showed two patterns: relatively slow in adult-onset subjects, and faster in juvenile-onset subjects. The progression rate seemed linear. Two patients with onset before age 14 became wheelchair-bound before age 28. A boy with onset at age 1 year needed assistance walking by age 12, and his February (2 of 2) 2001 NEUROLOGY 56 451 81 August 8th, 2000 Figure 2. Patient showing lordosis, scapular winging, proximal wasting affecting the pelvic and shoulder muscles, and a sparing of the facial muscles. mother’s help with most everyday tasks (Vignos’ scale grade 6). Mean CK was 589, ranging between 47 and 2,920 (normal 150 U/L). Normal CK was recorded in 40.6% of patients. Mean ASAT and ALAT were 35.7 and 44.5 U/L. Electromyography (EMG) showed myopathic changes with short duration, polyphasia, and low-amplitude poten- tials, which were more pronounced in the proximal muscles. Sensory and motor nerve conduction velocities were normal. Brain MRI, ECG, and echocardiograms, when performed, were normal. The inheritance pattern is consistent with autosomal dominant transmission; 44 of 76 (58%) children of affected parents manifested the disease. We identified 26 affected parent/affected child pairs. The pair-wise data comprises 18 pairs with a generation III parent and eight pairs with a generation IV parent. In the generation III parent pairs, we observed a mean decrease of 28.5 years in an offspring’s onset age. In the generation IV parent pairs, the mean decrease in an offspring’s onset age was 13.2 years. Thus, apparent anticipation was significant (p 0.000 in generation III parents and p 0.002 in generation IV parents). Overall comparison of age at onset curves in life table analysis between patients of generations III, IV, and V showed a decrease in age at onset (Wilcoxon test statistic 16.84, p 0.0002). We also examined the effects of parental gender origin on anticipation. The mean difference in age at onset between parent and child in 17 mother/child pairs was 1.6 years younger than in 9 father/child pairs (two-tailed Mann–Whitney U test 59.5, p 0.367). In our data, no evidence suggests a significant effect of parental gender on age at onset. Muscle biopsy. Light microscopy. Open muscle biopsy was performed on patients III-8, IV-6, IV-11, IV-21, and V-11. When biopsied, their ages varied between 9 and 59 years (mean 31.4 years). Morphologic findings were similar in all cases, composed of abnormal fiber size and shape variation, increased endo- and perimysial connective tissue, scattered degenerative fibers with myophagia, abnormal Z-bands, and, in three of five cases, prominent rimmed vacuoles (figure 3A). Central nuclei were occasionally present. Fiber type differentiation and distribution were normal. One patient (V-11) showed a significant number of ragged-red fibers (RRF) (15%); cytochrome c oxidase (COX)–succinate dehydrogenase technique disclosed between 5 and 30% COX-negative fibers in three of five cases. All RRF were COX-negative, but COX-negative fibers without signs of mitochondrial proliferation were also present. Immunohistochemical stains for dystrophin and sarcoglycans were normal. No abnormal deposits of tau, ubiquitin, or -amyloid proteins were found. Desmin was overexpressed in some fibers, but was not abundant enough for consideration as a significantly abnormal desmin accumulation. Vimentin and dystrophin-related protein overexpression in scattered small fibers without Figure 3. (A) Muscle cryostat section in Subject IV-6. Notice increased fiber size variability, one small fiber with two prominent rimmed vacuoles (bottom right corner) and a fiber with subsarcolemmal basophilia and marked intermyofibrillary network, indicative of mitochondrial proliferation (center) (hematoxylin– eosin 60 before reduction). (B) Electron micrograph in Subject V-11. Skeletal muscle fiber cut transversally. Notice increased number of paranuclear mitochondria with abnormal cristae and paracrystalline inclusions (8,000 before reduction). 452 NEUROLOGY 56 February (2 of 2) 2001 82 August 8th, 2000 dystrophin deficiency was considered evidence of regeneration. Electron microscopy. Three cases showed autophagic vacuoles with prominent pseudomyelin figures and dense bodies. One case (IV-6) showed intracytoplasmic bundles of 16- to 18-nm filaments. Despite thorough searching, no additional filamentous inclusions were observed in the nuclei or in the other four patients’ cytoplasm or nuclei. One patient (V-11) showed prominent subsarcolemmal and paranuclear abnormal mitochondria accumulations, many with paracrystalline inclusions (see figure 3B). Linkage analysis. Using genotype data of SSR markers, three-point analyses excluded linkage of the LGMD in our pedigree to known chromosomal loci for the following: LGMD forms 1A, 1B, 1C, 1D, and 1E, autosomal dominant and recessive HIBM, Bethlem myopathy, autosomal dominant Emery–Dreifuss dystrophy (AD-EDMD), and FSHD based on three-point lod scores 2.0. Discussion. Thirty-two patients were identified from a large Spanish family with autosomal dominant muscular dystrophy. The disorder fulfills LGMD diagnostic criteria,21 with the following clinical features: slowly progressive proximal symmetric weakness with predominantly lower limb onset, normal to mildly raised CK activity, myopathic EMG and muscle biopsy changes, and normal skeletal muscle dystrophy staining. Variability was observed in age at onset, muscular symptomatology, and progression rates, with two patient groups differentiated by severity and progression: an adult-onset form, around the fourth decade, and a juvenile form, beginning before age 15 years. Patients with severe functional disability belonged to the second group. Dysarthria, cardiac involvement, calf hypertrophy, and contractures are features of other ADLGMD forms, not seen in our patients. Another feature of our family was an early onset age (mean 16.3 years). In nearly two thirds of patients, onset occurred in childhood or adolescence. Patients with onset before 12 years of age presented with generalized muscular weakness. We noted an anticipation phenomenon in 26 parent– child pairs, with the parents two and three generations removed from a common ancestor. Earlier onset in children than in parents suggested genetic anticipation. Disease severity seems unrelated to the transmitting parent’s gender. Anticipation phenomenon was described in LGMD-1.6 The absence of facial, bulbar, or cardiac impairment excluded other myopathies. The lack of early joint contractures suggests our AD-LGMD pedigree differs from Bethlem myopathy or AD-EDMD. We excluded linkage to chromosomal loci for Bethlem myopathy and AD-EDMD.17,18,22-24 Some patients with FSHD lack facial involvement, thus resembling LGMD. However, in our family the absence of shoulder girdle muscle weakness asymmetry, predominant muscle weakness of scapular fixators, hearing loss, and facial weakness affecting eye closure and perioral muscles makes FSHD un- likely. Onset before 5 years of age is rare in FSHD, but was observed in our pedigree. We excluded linkage of the diseases to the 4q35 FSHD locus.19 Muscle biopsies showed rimmed vacuoles in three patients. Initially, a diagnosis of HIBM was considered because of this and the presence of filamentous inclusions.25 Most HIBM cases reported have shown autosomal recessive inheritance26-27 but AD-HIBM has been rarely described.28-29 However, rimmed vacuoles without the characteristic filaments are frequent nonspecific findings in different muscle disorders, including forms of LGMD17,30 and primary dystrophinopathies.31 We excluded linkage to the identified loci for the identified loci of autosomal recessive and dominant HIBM.15-16 We interpreted the mitochondrial proliferation in one patient’s muscle as a secondary phenomenon. Five AD-LGMD (LGMD1) forms have so far been delineated.2,5,8-12 Types 1B and 1E are associated with cardiologic abnormalities, including atrioventricular conduction disturbances, arrhythmias and sudden death. Type 1A is characterized by a dysarthric speech pattern not observed in our patients. LGMD 1C is caused by caveolin-3 mutations.9-10 LGMD 1D has been mapped to chromosome 6q23.11,12 Our linkage-analysis data using markers for those loci found no association, suggesting a genetically distinct disorder. We are performing a genome-wide scan to identify the disease locus. References 1. Bushby KMD, Beckmann JS. Workshop Report: the limbgirdle muscular dystrophies—proposal for a new nomenclature. 30th and 31st ENMC International Workshops, Naarden, the Netherlands, 6 — 8 January, 1995. Neuromuscular Disord 1995;5:337–343. 2. Beckmann JS, Brown RH, Muntoni F, et al 66th/67th ENMC sponsored international workshop: the limb-girdle muscular dystrophies, 26 –28 March 1999, Naarden, the Netherlands. Neuromuscul Disord 1999;9:436 – 445. 3. Bushby KMD. Making sense of the limb-girdle muscular dystrophies. Brain 1999;122:1403–1420. 4. Gilchrist JM, Pericak–Vance M, Silverman L, et al. Clinical and genetic investigation in autosomal dominant limb-girdle muscular dystrophy. Neurology 1988;38:5–9. 5. Speer MC, Yamaoka LH, Gilchrist JH, et al. Confirmation of genetic heterogeneity in limb-girdle muscular dystrophy: linkage of an autosomal dominant form to chromosome 5q. Am J Hum Genet 1992;50:1211–1217. 6. Speer MC, Gilchrist JM, Stajich JM, et al. Evidence for anticipation in autosomal dominant limb-girdle muscular dystrophy. J Med Genet 1998;35:305–308. 7. van der Kooi AJ, Ledderhof TM, de Voogt WG, et al. A newly recognized autosomal dominant limb girdle muscular dystrophy with cardiac involvement. Ann Neurol 1996;39:636 – 642. 8. van der Kooi AJ, van Meegen M, Ledderhof TM, et al. Genetic localization of a newly recognized autosomal dominant limbgirdle muscular dystrophy with cardiac involvement (LGMD1B) to chromosome 1q11–21. Am J Hum Genet 1997; 60:891– 895. 9. Minetti C, Sotgia F, Bruno C, et al. Mutations in the caveolin-3 gene cause autosomal dominant limb-girdle muscular dystrophy. Nat Genet 1998;18:365–368. 10. McNally EM, de Sa Moreira E, Duggan DJ, et al. Caveolin-3 in muscular dystrophy. Hum Mol Genet 1998;7:871– 877. 11. Messina DN, Speer MC, Pericak–Vance MA, et al. Linkage of familial dilated cardiomyopathy with conduction defect and February (2 of 2) 2001 NEUROLOGY 56 453 83 August 8th, 2000 12. 13. 14. 15. 16. 17. 18. 19. 20. muscular dystrophy to chromosome 6q23. Am J Hum Genet 1997;61:909 –917. Speer MC, Vance JM, Grubber JM, et al. Identification of a new autosomal dominant limb-girdle muscular dystrophy locus on chromosome 7. Am J Hum Genet 1999;64:556 –562. Vignos PJ, Spencer GE, Archibald KC. Management of progressive muscular dystrophy in childhood. JAMA 1963;184: 89 –96. Brooke MH, Fenichel GM, Griggs RC, et al. Clinical investigation in Duchenne dystrophy. 2. Determination of the “power” of therapeutic trials based on the natural history. 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Diagnostic criteria for neuromuscular disorders. 2nd ed. London, UK: Royal Society of Medicine Press, 1997:17–22. 22. Jöbsis GJ, Boers JM, Barth PG, et al. Bethlem myopathy: a slowly progressive congenital muscular dystrophy with contractures. Brain 1999;112:649 – 655. 23. Bonne G, Di Barletta MR, Varnous S, et al. Mutations in the gene encoding lamin A/C cause autosomal dominant Emery– Dreifuss muscular dystrophy. Nat Genet 1999;21:285–288. 24. Felice KJ, Schwartz RC, Brown CA, et al. Autosomal dominant Emery-Dreifuss dystrophy due to mutations in rod domain of the lamin A/C gene. Neurology 2000;55:275–280. 25. Carpenter S. Inclusion body myositis, a review. J Neuropathol Exp Neurol 1996;55:1105–1114. 26. Neufeld MY, Sadeh M, Assa B, et al. Phenotypic heterogeneity in familial inclusion body myopathy. Muscle Nerve 1995;18: 546 –548. 27. Argov Z, Tiram E, Eisenberg I, et al. Various types of hereditary inclusion body myopathies map to chromosome 9p1-q1. Ann Neurol 1997;41:548 –551. 28. Klingman JG, Gibbs MA, Creek W. Familial inclusion body myositis. Neurology 1991;41(suppl 1):275. Abstract. 29. Neville HE, Baumbach LL, Ringel SP, et al. Familial inclusion body myositis: evidence for autosomal dominant inheritance. Neurology 1992;42:897–902. 30. Marconi G, Pizzi A, Arimondi CG, et al. Limb girdle muscular dystrophy with autosomal dominant inheritance. Acta Neurol Scand 1991;83:234 –238. 31. de Visser M, Bakker E, Defesche JC, et al. An unusual variant of Becker muscular dystrophy. Ann Neurol 1990;27:578 –581. 454 NEUROLOGY 56 February (2 of 2) 2001 84 Ayúdanos a caminar, ayúdanos a vivir. ¡Juntos podemos conseguirlo! Paraje de Reolid 2, 30420 Valentín, Murcia, España C.F.G73886483 Email: [email protected] Web: www.conquistandoescalones.org Facebook: www.facebook.com/conquistandoescalones Twitter: @AceDistrofia