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A S S O C I A Z I O N E

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INDICE• Cos'è A.C.E.?.............................................................................................................. 4

• Riassunto e traduzione delle pubblicazioni...............................................................10

• Pubblicazioni scientifiche.......................................................................................... 21

• Incomplete penetrance in limb-girdle muscular dystrophy type 1F.....................22Marina 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..................24Vincenzo Nigro e Marco Savarese

• P.5.10 - Clinical and ultrastructural changes in transportinopathy......................36C. Angelini 1, E. Peterle 1, M. Fanin 1, G. Cenacchi 2, V. Nigro 3

• P.5.12 - A mutation in TNPO3 causes LGMD1F and characteristic nuclear pathology............................................................................................................. 37A. 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..38

• Next-Generation Sequencing Identifies Transportin 3 as the Causative Gene for LGMD1F.............................................................................................................. 39Annalaura Torella1,2, Marina Fanin3, Margherita Mutarelli1, Enrico Peterle3, Francesca DelVecchio Blanco2, Rossella Rispoli1,4, Marco Savarese1,2, Arcomaria Garofalo2, GiulioPiluso2, Lucia Morandi5, Giulia Ricci6, Gabriele Siciliano6, Corrado Angelini3,7, VincenzoNigro1,2

• Clinical phenotype, muscle MRI and muscle pathology of LGMD1F..................46Enrico Peterle1 , Marina Fanin1 , Claudio Semplicini1 , Juan Jesus Vilchez Padilla2 ,Vincenzo Nigro3,4 , Corrado Angelini1,5

• Limb-girdle muscular dystrophy 1F is caused by a microdeletion in the transportin 3 gene................................................................................................55Maria J. Melià1,2, Akatsuki Kubota3, Saida Ortolano4, Juan J. Vilchez5, Josep Gámez6,Kurenai Tanji7, Eduardo Bonilla3,7,†, Lluis Palenzuela1,2, Israel Fernandez-Cadenas1, AnnaPristoupilová8,9, Elena Garcia-Arumí1,2, Antoni L. Andreu1,2, Carmen Navarro2,4, MichioHirano3,and Ramon Marti1,2

• P07 Limb-Girdle Muscular Dystrophy and Inherited Myopathy Limb Girdle Muscular dystrophy 1F: Clinical, Molecular and Ultrastructural study (P07.032)............................................................................................................................. 65Corrado Angelini1, Enrico Peterle2, Marina Fanin3, Giovanna Cenacchi4 and VincenzoNigro5

• Ultrastructural changes in LGMD1F.................................................................... 66Giovanna Cenacchi1, Enrico Peterle2, Marina Fanin2, Valentina Papa1, Roberta Salaroli1

and Corrado Angelini2,3

• D.O.3 Next generation sequencing application are ready for genetic diagnosis ofmuscular dystrophies...........................................................................................71M. 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

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• New quantitative MRI indexes useful to investigate muscle disease..................72C. Angelini, M. Fanin, E. Peterle

• Identificazione di nuovi geni coinvolti nelle distrofie muscolari dei cingoli mediante arrays e sequenziamento di nuova generazione (NGS).....................73A. Torella 1, F. Del Vecchio Blanco 3, M. Dionisi 2, A. Garofalo 3, M. Iacomino 3, M. Mutarelli2, M. Savarese 1, G. Piluso 1, V. Nigro 1

• LGMD 1(F) - A pathogenetic hypotesis based on histopathology and ultrastructure........................................................................................................74G. Cenacchi, E. Peterle, L. Tarantino, V. Papa, M. Fanin, C. Angelini

• A novel autosomal dominant limb-girdle muscular dystrophy (LGMD 1F) maps to7q32.1-32.2..........................................................................................................75L.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..........................................78J. 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

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Che cos’è A.C.E.?

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1. L’ASSOCIAZIONE

- CHI SIAMO

ACE – Associazione Conquistando Escalones

Associazione senza scopo di lucro, fondata da malati di DISTROFIA MUSCOLARE DEI CINGOLI 1F – LGMD1F, loro familiari ed amici.

- DA DOVE VENIAMO

La nostra malattia colpisce la nostra vita quotidiana provocando pesanti limitazioni. È degenerativa, inizia colpendo le nostre capacità motorie e finisce compromettendo la nostra capacità respiratoria e la funzione cardiaca, portandoci alla morte. Inoltre, si tratta di una patologia ereditaria di cui sono state identificate già più di 8 generazioni di malati.

- DOVE ANDIAMO

Per questo motivo e per il fatto che la ricerca ha fatto molti passi avanti, abbiamo fondato l’associazione, dato che abbiamo bisogno di raccogliere fondi per portare a termine le ricerche in corso e trovare una cura per questa malattia ed altre malattie neuromuscolari.

- IL NOSTRO NOME

Il nome dell’Associazione è in spagnolo, in quanto la maggioranza dei malati vive in Spagna, ma il significato è facilmente comprensibile: “Conquistando Scalini”. L’idea deriva dal fatto che la vita di noi malati di Distrofia Muscolare, tra tante altre cose, è segnata dagli scalini. Uno dei primi sintomi, infatti, è quello di fare fatica a salire le scale. Il decorso della malattia è caratterizzato da un processo degenerativo che porta alla sedia a rotelle, ed infine, quando colpisce gli organi interni, alla morte.

Noi però abbiamo scelto il nome “Conquistando Escalones” perché il messaggio che vogliamo dare è positivo, e cioè che passo dopo passo, scalino dopo scalino, conquisteremo la vetta della montagna, trovando la cura per la nostra malattia e per molte altre patologie simili.

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2. LA NOSTRA PATOLOGIA

Nell’immaginario collettivo, se una malattia non ha dei sintomi così evidenti, sembra che non sia poi così grave e noi spesso siamo costretti a scontrarci con questa giudicante indifferenza. Fino a quando la patologia non è agli stadi finali, infatti, può capitare che ad un primo sguardo veloce o poco esperto non ci si renda conto che siamo colpiti da una disabilità fisica che compromette pesantemente la nostra salute e la nostra vita.

Le difficoltà iniziano da bambino, quando vedi che i tuoi amici corrono, saltano, giocano a nascondino, si divertono e tu non puoi far altro che stare in un angolino a guardare. La tua testa ti dice di andare a giocare e divertirti con loro, ma i tuoi muscoli non sono d’accordo e te lo impediscono.

Piano piano inizi a fare fatica a fare le scale; ti aggrappi a quel passamano, senza il quale non ce la faresti, ma un giorno anche lui ti abbandona e hai bisogno di braccia forti che ti sorreggano e ti accompagnino lungo tutti gli scalini che la vita ti mette di fronte.

Inciampi, cadi e resti lì per terra, in attesa che qualcuno ti veda e ti rialzi... Il tuo corpo tornerà in piedi, ma la tua anima, caduta dopo caduta, farà sempre più fatica a rialzarsi.

Cadi una, due, mille volte, fino a quando non puoi fare a meno di sederti su una sedia a rotelle e guardare la vita da un’altra prospettiva.

Inizia a mancarti il respiro, fai molta difficoltà a deglutire, ti rendi conto che il tuo corpo non riesce più a stare dietro alla tua mente e dimentichi anche cosa voglia dire poterti pettinare i capelli, vestirti, mangiare, andare in bagno, lavarti da solo. Non hai più la tua autonomia e la tua libertà.

Il tempo inizi a vederlo come un muro che ti corre incontro dal quale non hai scampo. Hai la fortuna di vedere attorno a te parenti malati che stanno ancora molto meglio di te, ma purtroppo anche la sfortuna di vedere come altri piano piano ti salutano e ti lasciano per sempre. E se c’è qualcosa di più difficile del vedere la tua famiglia e i tuoi amici che si spengono e ti lasciano a causa di questa malattia, è avere la cura a portata di mano e non essere in grado di raggiungerla a causa della mancanza di fondi.

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3. RICERCA

- DOVE SIAMO

Vi illustriamo brevemente quelli che sono i risultati raggiunti grazie alla ricerca negli ultimi anni:

Nel 2013 è stato scoperto il gene che, codificando erroneamente la proteina Trasportina 3, causa la Distrofia Muscolare dei Cingoli 1F - LGMD1F. Importanti risultati della ricerca hanno evidenziato come la stessa proteina sia coinvolta nella trasmissione del virus dell’AIDS e come vi siano molte similitudini tra questa Distrofia ed altre patologie neuromuscolari rare. Questo comporta che gli studi di ricerca compiuti sulla nostra Distrofia Muscolare, in realtà, diano contemporaneamente dei contributi su scala mondiale, anche nella ricerca su una possibile terapia per l’AIDS e su numerose altre patologie neuromuscolari che coinvolgono milioni di persone in tutto il mondo.

- RELAZIONE DELLA NOSTRA RICERCA CON QUELLA DELL’AIDS

Come abbiamo detto prima, dopo la scoperta del gene si è visto che esiste una mutazione che colpisce la Trasportina 3. È stata una sorpresa perché la suddetta Trasportina si conosce da anni come la proteina chiave nell’infezione delle cellule immunitarie da parte dell’AIDS, ma non si era mai pensata come molecola bersaglio per la sua importanza in questi processi.

In base a quanto spiegano articoli scientifici pubblicati:

le trasportine fungono da tassista tra il nucleo cellulare e il citoplasma, bisogna tener conto che il nucleo è completamente isolato dal resto della cellula e c’è solo una via d’entrata e uscita che sono i pori nucleari. Tuttavia, le molecole non possono passare liberamente attraverso questi, devono essere accompagnate e, in questo caso, queste accompagnatrici sono le trasportine.

Tra altre funzioni, le trasportine sono le incaricate di portare gli RNA messaggeri dal nucleo fino al citoplasma dove vengono tradotti e, a sua volta, si occupano di riportare al nucleo certe proteine come fattori di trascrizione.

Inoltre spiegano che:

La scoperta della mutazione nella nostra famiglia ha dimostrato che, anche se la Trasportina è mutata, c’è vitalità cellulare e le prove hanno dimostrato

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che la mutazione di questa proteina nelle cellule immunitarie frena l’infezione da AIDS, per cui la chiave sarebbe sviluppare un trattamento che colpisca la Trasportina 3, ma solo nelle cellule immunitarie, per evitare gli effetti della distrofia muscolare. Questo permetterebbe, senza dubbio, una buona terapia nei confronti dell’AIDS, prevenendo da un lato la malattia e, dall’altro, evitando che si moltiplichi in persone già colpite.

Qualcosa che si studia e si ricerca da anni è comparso in maniera naturale e spontanea nella nostra famiglia, una mutazione che non compromette la vitalità cellulare (tranne in alcune cellule muscolari) e che fornisce l’immunità.

Continuare questa linea di ricerca dipende principalmente dalla possibilità di ricevere i finanziamenti necessari. Da qui l’importanza di ottenere fondi e, da qui, la creazione della nostra associazione come strumento per raggiungere questo obiettivo. È di vitale importanza per noi controllare che i fondi ottenuti vengano destinati a finalizzare la ricerca e che ci portino al trattamento e la cura della Distrofia Muscolare dei Cingoli collaborando, inoltre, nel progresso medico-scientifico di altre malattie rare e dell’AIDS.

- LINEE DI RICERCA ATTUALI

Attualmente ci sono diverse linee di ricerca in tutto il mondo. Tra le più importanti segnaliamo quelle che sono in corso in Italia e in Spagna:

1. in Italia: Vincenzo Nigro mira a scoprire il meccanismo della proteina Trasportina 3, meccanismo patologico del tutto nuovo che potrebbe spiegare il funzionamento anche di altre malattie simili che colpiscono i muscoli.

2. in Spagna: abbiamo tre linee diverse, seguite da Juan Jesús Vilchez, José Alcamí e Rubén Artero, che collaborano nello studio di vari aspetti: da come silenziare il gene “difettoso”, a come si sviluppa e si riproduce questa particolare Distrofia dei Cingoli, fino all’implicazione che potrebbe avere il fatto che le persone affette da questa malattia sono potenzialmente immuni all’AIDS. Già dal 2013, a Madrid, José Alcamí ha avviato uno studio volto a capire il meccanismo della proteina che, nel nostro caso comporta la Distrofia Muscolare di cui siamo affetti, mentre nel caso dell’AIDS impedisce al virus dell’HIV di entrare nelle cellule e far insorgere tale Sindrome. Se si riuscisse a capire come disattivare il difetto genetico che la provoca, si riuscirebbe anche a trovare il modo di creare un vaccino per l’AIDS.

3. In un laboratorio in Belgio: Frauke Christ sta seguendo la medesima linea di ricerca di José Alcamí, collaborando con lo stesso, investigando però su diversi aspetti della malattia.

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Noi malati di questa Distrofia Muscolare stiamo già collaborando con questi studiosi da due anni, fornendo campioni di sangue e quanto necessario affinché questi laboratori siano agevolati nel loro lavoro.

Per proseguire e sviluppare queste ricerche c’è bisogno di un continuo apporto di finanziamenti, visto il gran numero di ricercatori e strumentazione necessari, trattandosi di meccanismi genetici completamente nuovi che aprirebbero le porte alla comprensione di molte patologie e porterebbero alla realizzazione di nuove tecniche di terapia genica.

La nostra Associazione ha un ruolo tutt’altro che marginale: oltre a promuovere la raccolta di fondi, si occupa di veicolare i finanziamenti ai laboratori di ricerca e di coordinare le linee investigative in atto, evitando doppioni negli studi che comporterebbero un inutile spreco di tempo e risorse. Concretamente, oltre a mettere in contatto tra loro i ricercatori, organizziamo lo scambio di materiale investigativo quale campioni di sangue o biologici, affinché i vari laboratori possano sempre contare su informazioni il più aggiornate possibili.

Siamo in contatto diretto costante con molti dei laboratori che stanno effettuando ricerche su di noi, per monitorare gli investimenti fatti e i relativi progressi ottenuti.

La nostra Associazione è continuamente alla ricerca di fondi e visibilità per poter raggiungere l’obbiettivo per cui è nata: avere la possibilità di proseguire gli studi per poter dare un futuro migliore a milioni di persone.

La spinta per costituire questa Associazione ce l’ha data vedere che grazie alla benevolenza di alcuni medici e ricercatori, che a loro volta hanno invogliato e coinvolto altri laboratori e medici che fino a poco tempo fa non sapevano nemmeno della nostra esistenza, si stanno facendo passi avanti nella conoscenza della nostra malattia e di una possibile terapia futura. Ma a causa della scarsità di fondi, visto anche il momento storico che stiamo attraversando, tutto questo potrebbe essere mandato in fumo.

Abbiamo bisogno dell’impegno concreto di tutti!



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Riassunto e traduzione delle pubblicazioni

11 A S S O C I A Z I O N E

Dicembre 2014

Lettera all’editore. Penetranza Incompleta nella Distrofia Muscolare deiCingoli 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. Dipartimento di Neuroscienze, Università di Padova, Padova, Italia 2. IRCCS Fondazione San Camillo, Venezia, Italia 3. Dipartimento di Biochimica, Biofisica e Patologia Generale, II Università di Napoli, Napoli, Italia 4. Istituto Telethon di Genetica e Medicina (TIGEM), Napoli, Italia

La Distrofia Muscolare dei Cingoli (LGMD) tipo 1F (MIM #608423) è una rara patologiaautosomica dominante il cui locus è stato mappato ed è stato identificato il gene aseguito di una ricerca in una stessa numerosa famiglia. Durante l’esame di questafamiglia, si è ampliata la genealogia iniziale e le caratteristiche cliniche della malattia. Sicaratterizza per un grado variabile di debolezza muscolare e limitazione funzionale, conun esordio dei sintomi o prima dei 15 anni (forma giovanile) o nella decade dai 30 ai 40(forma adulta). La ricerca clinica genetica in questa famiglia ha rivelato che, in alcunipazienti, la malattia si trasmette attraverso genitori apparentemente non colpiti(penetranza incompleta). Per calcolare il tasso di penetranza esatto, si è esaminatotanto il fenotipo clinico quanto il genotipo di 115 membri della famiglia. I risultati ottenutipossono essere utili per una consulenza genetica, specialmente per i pazienti piùgiovani in cui la mutazione è presente.

Maggio 2014

Acta Miologica 2014; XXXIII: p. 1-12.Base genetica della distrofia muscolare deicingoli: aggiornamento al 2014

Vincenzo Nigro e Marco Savarese

Dipartimento di Biochimica, Biofisica e Patologia Generale, Seconda Università degli Studi di Napoli eIstituto Telethon di Genetica e Medicina (TIGEM), Napoli, Italia

Le distrofie muscolari dei cingoli (LGMD) sono un gruppo di alterazioni muscolarialtamente eterogenee, che inizialmente colpiscono i muscoli volontari dell’area deicingoli pelvico e scapolare. La definizione è molto descrittiva e meno ambigua peresclusione: non X linked, non FSH, non miotonica, non distale, non sindromica e noncongenita. Attualmente, la classificazione genetica sta diventando troppo complessadato che l’acronimo LGMD è stato utilizzato anche per altre patologie muscolari confenotipi sovrapposti.Attualmente, la lista di geni da rivedere è troppo vasta per un approccio gene per geneed è più adatta per essere mirata a pannelli di Next Generation Sequencing (NGS) chedovrebbero includere qualsiasi gene che fino adesso sia stato associato al quadroclinico di LGMD.Il presente articolo ha lo scopo di riassumere la base genetica delle LGMD ordinando eproponendo una nomenclatura per le forme orfane. Questo è utile visto il ritmo dellenuove scoperte.Fino ad oggi si sono identificati trentun loci, 8 autosomici dominanti e 23 autosomicirecessivi. Le forme dominanti (LGMD1) sono: LGMD1A (myotilin), LGMD1B (lamin A/C),LGMD1C (caveolin3), LGMD1D (DNAJB6), LGMD1E (desmin),LGMD1F (transportin3), LGMD1G (HNRPDL), LGMD1H (chr. 3). Le forme autosomiche recessive (LGMD2)sono LGMD2A (calpain3), 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 (anoctamin5), LGMD2M (fukutin), LGMD2N (POMT2), LGMD2O (POMTnG1),LGMD2P (dystroglycan), LGMD2Q (plectin), LGMD2R (desmin), LGMD2S(TRAPPC11), LGMD2T (GMPPB), LGMD2U (ISPD), LGMD2V (Glucosidase, alpha ),LGMD2W (PINCH2).

LGMD autosomiche dominanti

LGMD1F è stata mappata originariamente in un intervallo di 3.68 Mb nel cromosoma7q32.1-7q32.2 in una numerosa famiglia italo-spagnola. Abbiamo presentatol’identificazione di TNPO3 mediante un sequenziamento esomico completo di 4 membrimalati della famiglia e il completo perfezionamento della regione al WMS 2012. I datisono stati quindi pubblicati: una mutazione nella fase di lettura nel gene dellatrasportina 3 (TNPO3) è condivisa da tutti i membri malati della famiglia con un 94% dipenetranza. Il gene TNPO3 è composto da 23 esoni e codifica una proteina di 923amminoacidi, espressa anche nel muscolo scheletrico. La proteina con la mutazionenella fase di lettura TNPO3 è più grande di quella del ceppo selvatico, dato che lemanca il suddetto stop codon e si trova attorno al nucleo ma non dentro. I pazienti conun esordio nell'adolescenza mostrano un fenotipo più severo con una progressionerapida, mentre i pazienti con un esordio in età adulta presentano una progressione piùlenta. Hanno un’atrofia marcata dei muscoli del cingolo pelvico, coinvolgendospecialmente il vastus lateralis e l’ileopsoas. È interessante che alcuni pazientipresentano disfagia, aracnodattilia e insufficienza respiratoria. Il range del CK è di 1–3volte. Non si è riscontrato coinvolgimento cardiaco.

RingraziamentiQuesto studio è stato supportato principalmente dai fondi di Telethon, Italia (TGM11Z06 to V.N.) eTelethon- UILDM (Unione Italiana Lotta alla Distrofia Muscolare) (GUP 10006 and GUP11006 to V.N.). Ifinanziatori non hanno avuto nessun ruolo nello studio, raccolta ed analisi dei dati, decisione sullapubblicazione o preparazione del manoscritto.

Ottobre 2013

P.5.10 - Cambiamenti clinici ed ultrastrutturali nella trasportinopatia

C. Angelini 1, E. Peterle 1, M. Fanin 1, G. Cenacchi 2, V. Nigro 3

1. Università di Padova, Padova, Italia; 2. Università di Bologna, Bologna, Italia; 3. Istituto Telethon di Genetica e Medicina TIGEM, Napoli, Italia

Si sono studiate in 3 biopsie le caratteristiche muscolari istopatologiche, ultrastrutturali egenetiche di una numerosa famiglia italo-spagnola con LGMD autosomica dominante,precedentemente mappata in 7q32.1–32.2 (LGMD1F).Abbiamo raccolto le cartelle cliniche in 19 di 60 pazienti; in un paio di malati si èstudiata l’analisi istopatologica delle biopsie muscolari (madre 1 biopsia, sua figlia 2biopsie consecutive ai 9 e 22 anni). Si è osservato che l’età d’esordio variava dai 2 ai 35anni e si è verificata tanto nel cingolo pelvico che in quello scapolare. In 14 casi si èriscontrata ipotrofia tanto nei muscoli prossimali superiori quanto nelle estremità inferiorinei polpacci. La gravità non è aumentata nelle successive generazioni. Conclusionicliniche non precedentemente notificate sono aracnodattilia, disfagia e disartria.Inoltre, abbiamo riscontrato discrepanza tra la gravità clinica e la biopsia muscolare: lafiglia ha un decorso clinico più grave, nella prima biopsia aveva unicamente atrofia dellefibre tipo 1, mentre l’atrofia delle fibre è aumentata nella seconda biopsia. La madreaveva una istopatologia del muscolo più compromessa (più variabilità delle fibremuscolari e cambiamenti autofagici con macchie di fosfatasi acida). La causa


dell’atrofia progressiva e la perdita di miofibrille è un assemblaggio sarcomericoanormale. Mediante microscopio elettronico si è individuato un accumulo di corpimiofibrillari nelle fibre muscolari. Si è osservato un accumulo di desmina e miotilina eaggregati p62.Si è scoperto come causa di questa malattia un difetto nel gene della trasportina 3 cherappresenta un nuovo meccanismo di miopatia dominante. I nostri dati morfologici eultrastrutturali sembrano seguire un fenotipo simile alle malattie miofibrillari; tuttavia,erano presenti anche autofagosomi. È possibile che le proteine SR non possanomigrare o essere trasportate fuori e dentro dalla membrana nucleare.

Ottobre 2013

P.5.12 - Una mutazione nel TNPO3 causa LGMD1F e una patologia nuclearecaratteristica

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, Dipartimento di Neurologia, New York, USA; 2. Vall d'Hebron Istituto di Ricerca, Università Autonoma di Barcellona, Gruppo di Ricerca sulle

Malattie Neuromuscolari e Mitocondriali, Barcellona, Spagna;3. Istituto di Ricerca Biomedica di Vigo (IBIV), Ospedale Universitario di Vigo (CHUVI), Dipartimento

di Patologia e Neuropatologia Vigo, Spagna; 4. Ospedale Universitario e Politecnico La Fe, Dipartimento di Neurologia, Valencia, Spagna;5. Ospedale Universitario Vall d'Hebron, Istituto di Ricerca, Università Autonoma di Barcellona,

Clinica per Malattie Neuromuscolari, Dipartimento di Neurologia, Barcellona, Spagna;6. Columbia University Medical Centre, Dipartimento di Patologia e Biologia Cellulare, New York,

USA;7. Centro Nazionale di Analisi Genomica, Barcellona, Spagna

La Distrofia Muscolare dei Cingoli 1F (LGMD1F) è una patologia autosomica dominanteche colpisce una famiglia spagnola. Usando un sequenziamento genomico completo, siè identificata la delezione di un unico nucleotide (c.2771del) nel gene della trasportina 3in un paziente con LGMD1F. La mutazione interrompe il codone di stop di TNPO3 ecausa una mutazione nella fase di lettura. La trasportina 3 è una proteina nucleare emedia l’importazione delle proteine ricche in serina-arginina al nucleo, che sonoimportanti per lo splicing del mRNA. L’oggetto dello studio è l’analisi della trasportina 3nella patogenesi della LGMD1F.Si è eseguito un sequenziamento del TNPO3 mediante dideossi in 24 pazienti malati e23 familiari sani. I campioni muscolari di 4 pazienti sono stati analizzati mediante metodiconvenzionali e immunoistochimica. Un sequenziamento diretto di TNPO3 ha mostratoche tutti i pazienti avevano una mutazione eterozigote e nessuno dei familiari saniaveva la mutazione. La colorazione del muscolo con ematossilina ed eosina (HE)hanno rivelato nuclei (10.7 ± 3.0%; media ± SD) con pallore centrale in tutti i pazientistudiati.La immunistochimica con anticorpi antitrasportina 3 mostrano una colocalizzazione coni nuclei nei soggetti di controllo. Nei pazienti, si è anche osservata la trasportina 3 nelnucleo, ma spesso disegualmente distribuita nella periferia, in un pattern di colorazionea macchie simile a quello osservato con HE. Gli studi genetici e istologici in una famigliaspagnola sostengono fortemente l’ipotesi che il gene TNPO3 è la causa genetica dellaLGMD1F. Gli studi patologici indicano anche che la distribuzione subcellulare dellatrasportina 3 è interrotta e colpisce la struttura dei nuclei.


Maggio 2013

Sequenziamento di ultima generazione identifica la Trasportina 3 come causagenetica di LGMD1F

Annalaura Torella1,2, Marina Fanin3, Margherita Mutarelli1, Enrico Peterle3, FrancescaDel Vecchio Blanco2, Rossella Rispoli1,4, Marco Savarese1,2, Arcomaria Garofalo2, GiulioPiluso2, Lucia Morandi5, Giulia Ricci6, Gabriele Siciliano6, Corrado Angelini3,7, VincenzoNigro1,2

1. TIGEM (Telethon Institute of Genetics and Medicine), Napoli, Italia,2. Dipartimento di Biochimica Biofisica e Patologia Generale, Seconda Università degli Studi di

Napoli, Napoli, Italia3. Dipartimento di Neuroscienze, Università degli Studi di Padova, Padova, Italia4. Cancer Research UK, Londra, Regno Unito5. Fondazione IRCCS Istituto Neurologico C. Besta, Milano, Italia6. Dipartimento di Medicina Clinica e Sperimentale, Università degli Studi di Pisa, Pisa, Italia7. IRCSS S. Camillo, Venezia, Italia

RiassuntoLe Distrofie Muscolari dei Cingoli (LGMD) hanno condizioni genetiche e clinicheeterogenee. Abbiamo studiato una numerosa famiglia con un pattern di trasmissioneautosomica dominante, precedentemente classificata come LGMD1F e mappato nelcromosoma 7q32.I membri malati si caratterizzano per debolezza muscolare che colpisce prima il cingolopelvico e l’ileopsoas.Abbiamo sequenziato l’esoma completo di 4 membri della famiglia ed abbiamoidentificato una variante condivisa della mutazione eterozigote della fase di lettura nelgene trasportina 3 (TNPO3), che codifica un membro della superfamiglia importina-β. Ilgene TNPO3 è mappato nell’intervallo critico della LGMD1F e il prodotto genico umanodei suoi 923 amminoacidi è espresso anche nel muscolo scheletrico. Inoltre, abbiamoidentificato un caso isolato di LGMD con una nuova mutazione missenso nello stessogene. Abbiamo localizzato il TNPO3 mutante attorno al nucleo, ma non dentro. Ilcoinvolgimento genetico connesso con il trasporto al nucleo suggerisce un nuovomeccanismo patologico che conduce alla distrofia muscolare.

Aprile 2013

Fenotipo clinico, MRI muscolare e patologia muscolare di LGMD1F

Enrico Peterle1 • Marina Fanin1 • Claudio Semplicini1 • Juan Jesus Vilchez Padilla2 •Vincenzo Nigro3,4 • Corrado Angelini1,5

1. Dipartimento di Neuroscienze, Università di Padova, Campus Biomedico “Pietro d’Abano”,Padova Italia

2. Dipartimento di Neurologia, Ospedale Universitario e Politecnico La Fe, Valencia, Spagna 3. Seconda Università degli Studi di Napoli, Dipartimento di Patologia Generale, Napoli, Italia;4. Istituto Telethon di Genetica e Medicina (TIGEM), Napoli, Italia5. IRCCS Ospedale San Camillo, Venezia, Italia

RiassuntoDelle 7 diverse forme genetiche autosomiche dominanti di LGMD descritte fino ad oggi,solamente in 4 è stato identificato il gene che le causa (LGMD1A-1D). Descriviamo lecaratteristiche cliniche, istopatologiche e MRI muscolare di una numerosa famiglia italo-spagnola con LGMD1F che presenta debolezza nei muscoli prossimali degli arti e neimuscoli assiali. Abbiamo ottenuto dati clinici completi e classificato la progressione dellamalattia in 29 pazienti. Si è realizzata una MRI muscolare in 7 pazienti. Si sono studiate3 biopsie muscolari di 2 pazienti. I malati con un’età di esordio nelle prime decadi


presentano un fenotipo più severo con un decorso rapido della malattia, quelli conesordio da adulti presentano uno sviluppo più lento. La MRI muscolare mostraun’importante atrofia nei muscoli degli arti inferiori, specialmente nel vastus lateralis.Ampliare la popolazione dei pazienti ha permesso l’identificazione di caratteristicheprecedentemente non segnalate, includendo disfagia, aracnodattilia e insufficienzarespiratoria. Le biopsie muscolari mostrano atrofia diffusa delle fibre che evolve neltempo, cambiamenti miopatici cronici, aree basofile citoplasmatiche, autofagosomi eaggregati miofibrillari e proteine citoscheletriche. La LGMD1F si caratterizza per uncoinvolgimento selettivo dei muscoli degli arti e insufficienza respiratoria in faseavanzata e per diversi gradi di progressione clinica. Nuove caratteristiche cliniche sonoemerse dallo studio di ulteriori pazienti.L’impiego di sequenziatori di ultima generazione (NGS) in questa famiglia ha dato comerisultato la recente identificazione della trasportina 3 (TNPO3) come la causa geneticadella LGMD1F (13). Questi nuovi risultati sono cruciali per capire il nesso tra imeccanismi patogenetici e le caratteristiche cliniche.

Marzo 2013

La Distrofia Muscolare dei Cingoli 1F causata da una microdelezione nel geneTrasportina 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. Gruppo di Ricerca sulle Malattie Neuromuscolari e Mitocondriali, Vall d’Hebron Istituto di Ricerca,Università Autonoma di Barcellona, Barcellona, Spagna

2. Biomedical Network Research Centre on Rare Diseases (CIBERER), Istituto di Salute Carlos III,Madrid, Spagna

3. Dipartimento di Neurologia, Columbia University Medical Centre, New York, USA4. Dipartimento di Patologia e Neuropatologia, Istituto per la Ricerca Biomedica di Vigo (IBIV),

Ospedale Universitario di Vigo (CHUVI), Vigo, Spagna5. Dipartimento di Neurologia, Ospedale Universitario e Politecnico La Fe, Valencia, Spagna, e

Biomedical Network Research Centre on Neurodegenerative Disorders (CIBERNED), Istituto diSalute Carlos III, Madrid, Spagna

6. Clinica per le Malattie Neuromuscolari, Dipartimento di Neurologia, Ospedale Universitario Valld’Hebron, Istituto di Ricerca, Università Autonoma di Barcellona, Barcellona, Spagna

7. Dipartimento di Patologia e Biologia Cellulare, Columbia University Medical Centre, New York,USA

8. Centro Nazionale di Analisi Genomica, Barcellona, Spagna9. Istituto di patologie metaboliche ereditarie, Prima Facoltà di Medicina, Università Carlo di Praga,

Praga, Repubblica Ceca

Nel 2001, abbiamo rilevato il linkage genetico di una forma autosomica dominante didistrofia muscolare dei cingoli, la distrofia muscolare dei cingoli 1F, nel cromosoma7q32.1-32.2, ma l’identificazione del gene mutante era sfuggente. Adesso, usando unastrategia di sequenziamento dell’intero genoma, abbiamo identificato la causa dellamutazione della distrofia muscolare dei cingoli 1F, una delezione eterozigote di unsingolo nucleotide (c.2771del) nel codone di stop della Trasportina 3 (TNPO3). Questogene si colloca dentro la regione cromosomica connessa alla malattia e codifica unaproteina della membrana nucleare appartenente alla famiglia delle beta importine. IlTNPO3 trasporta proteine ricche di serina/arginina nel nucleo ed è stato identificatocome un fattore chiave nel processo di importazione dell’HIV nel nucleo. La mutazionegenera un'estensione di 15 amminoacidi nel terminale C della proteina, isolato con ilfenotipo clinico ed è assente nella base di dati della sequenza genomica e un gruppo di>200 alleli di controllo. Nel muscolo scheletrico degli individui malati, l’espressionedell’RNA messaggero mutante e le anomalie istologiche dei nuclei e TNPO3 indicanouna funzione alterata del TNPO3. I nostri risultati dimostrano che la mutazione del


TNPO3 è la causa della distrofia muscolare dei cingoli 1F, ampliando le nostreconoscenze sulle basi molecolari delle distrofie muscolari e rinforza l’importanza deidifetti delle proteine dell’involucro nucleare come causa di miopatie ereditarie.

Febbraio 2013

P07 Distrofie muscolari dei cingoli e miopatie ereditarie. Distrofia muscolaredei cingoli 1F: Studio delle alterazioni cliniche, molecolari e ultrastrutturali.(P07.032)

Corrado Angelini1, Enrico Peterle2, Marina Fanin3, Giovanna Cenacchi4 and VincenzoNigro5

1. Università di Neuroscienze di Padova, Italia2. Università di Neuroscienze di Padova, Italia3. Università di Neuroscienze di Padova, Italia4. Università di Patologia di Bologna, Italia5. Università di Patologia Generale di Napoli, Italia

CONTESTO: Le LGMD sono un gruppo eterogeneo di malattie genetiche condebolezza nei muscoli prossimali degli arti e/o distali. Ad oggi sono conosciute 8 formedi LGMD autosomiche dominanti. Il fenotipo clinico della LGMD1F è caratterizzato dauna notevole varietà, che va da un esordio precoce, con una progressione rapida esevera a forme meno aggressive. Le caratteristiche cliniche e morfologiche dei pazienticon LGMD1F non sono state ancora sufficientemente caratterizzate per suggerire unaspecifica eziologia.METODO: Abbiamo raccolto le cartelle cliniche in 19 di 60 pazienti e abbiamo ampliatol’albero genealogico; in un paio di malati (madre 1 biopsia, sua figlia 2 biopsieconsecutive ai 9 e 22 anni) si è studiata l’analisi istopatologica, l’immunoistochimica(desmina, miotilina, p62) e microscopia elettronica delle biopsie muscolari. Il DNA di 4pazienti è stato studiato con la piattaforma MotorChip CGH array per identificare il generesponsabile.RISULTATO: Si è osservato che l’età d’esordio variava dai 2 ai 35 anni; in metà dei casisi è riscontrata ipotrofia tanto nei muscoli prossimali superiori come nelle estremitàinferiori nei polpacci. Inoltre, abbiamo riscontrato discrepanza tra la gravità clinica e ilcoinvolgimento della biopsia muscolare: la figlia (caso di riferimento) ha un decorsoclinico più grave, e una maggiore atrofia delle fibre muscolari, invece la madre avevauna istopatologia del muscolo più compromessa (più variabilità delle fibre muscolari ecambiamenti autofagici con macchie di fosfatasi acida). Si è osservato un accumulo didesmina e miotilina e aggregati p62. Mediante microscopio elettronico si è individuatoun accumulo di corpi miofibrillari nelle fibre muscolari. La MRI muscolare nel paziente diriferimento mostra una severa e selettiva atrofia nel vastus lateralis.CONCLUSIONI: I nostri studi morfologici ed ultrastrutturali sembrano suggerire unamiopatia con fenotipo analogo a quelli descritti per malattie Z-disk. Anche se lo specificodifetto genetico non è ancora noto, è possibile ipotizzare che LGMD1F possa portare aduna scomposizione della rete citoscheletrica desmina correlata.

Con il sostegno di: Telethon Italia, AFM (Association Francaise contre les Myopathies).Informativa: Dr. Angelini ha ricevuto un compenso personale per le attività con Genzyme come mebro delComitato Consultivo. Dr. Peterle non ha nulla da comunicare. Dr. Fanin non ha nulla da comunicare. Dr.Cenacchi non ha nulla da comunicare. Dr. Nigro non ha nulla da comunicare.


Dicembre 2012

Alterazioni ultrastrutturali in LGMD1F

Giovanna Cenacchi1, Enrico Peterle2, Marina Fanin2, Valentina Papa1, Roberta Salaroli1and Corrado Angelini2,3

1. Dipartimento di Scienze Biomediche e Neuromotorie, “Alma Mater” Università di Bologna,Bologna,

2. Dipartimento of Neuroscienze, Università di Padova, Padova 3. IRCCS S. Camillo, Venezia, Italia

In una numerosa famiglia italospagnola con eredità autosomica dominante è statasegnalata debolezza muscolare dei muscoli prossimali degli arti e dei muscoli assiali.Sono state descritte le caratteristiche cliniche, genetiche e istologiche. È statoprecedentemente identificato il locus nel cromosoma 7q32.1-32.2 per questa distrofiamuscolare dei cingoli 1F (LGMD1F). Segnaliamo uno studio patologico muscolare di 2pazienti (madre e figlia) di questa famiglia. I risultati morfologici muscolari mostrano unincremento della variabilità della dimensione delle fibre, atrofia delle fibre e vacuolipositivi alla fosfatasi acida. L’immunofluorescenza per desmina, miotilina, p62 e LC3 hamostrato un accumulo di miofibrille, aggregati di proteine leganti l’ubiquitina eautofagosomi.Lo studio ultrastrutturale conferma i vacuoli autofagosomali.Si sono rilevate diverse alterazioni dei componenti miofibrillari, come un importantedisordine, strutture bastoncellari con aspetto granulare e occasionalmente corpicitoplasmatici. I nostri dati ultrastrutturali e le caratteristiche patologiche muscolari sonocaratteristiche della LGMD1F e sostengono l’ipotesi che i difetti genetici portano a unamiopatia fenotipica associata a un scomposizione della rete citoscheletrica.

I nostri dati morfologici e ultrastrutturali suggeriscono nei nostri casi di LGMD1F unamiopatia fenotipica simile a quelle descritte per le malattie Z-disk. Anche se i difettigenetici sono ancora in fase di studio, è possibile ipotizzare che la proteina mutante inLGMD1F possa provocare una scomposizione della rete citoscheletrica desminacorrelata.

Agosto 2012

D.O.3 - Le applicazioni del sequenziamento di ultima generazione sono pronteper diagnosi genetiche di distrofie muscolari

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 diPatologia Generale, Napoli, Italia;

2. TIGEM, Telethon Institute of Genetic and Medicine, Napoli, Italia;3. Seconda Università degli Studi di Napoli, Dipartimento di Patologia Generale, Napoli, Italia;4. Seconda Università degli Studi di Napoli, Cardiomiologia e Genetica Medica, Napoli, Italia;5. Università degli Studi di Padova, Dipartimento of Neuroscienze, Padova, Italia

Il sequenziamento di ultima generazione (NGS) sta avendo un forte impatto sulla nostraconoscenza dei diversi aspetti della biologia. Può essere inoltre molto potente perstudiare pazienti con condizioni genetiche eterogenee, come le distrofie muscolari. Inprimo luogo per identificare nuovi geni usando il risequenziamento dell’esoma.Secondariamente, per diagnosticare mutazioni in tutti i geni causativi conosciuti,quando utilizzati come approccio mirato. In terzo luogo per ottenere conoscenzasull’impatto delle mutazioni nell’espressione e splicing del mRNA nei muscoli malati.


Abbiamo utilizzato NGS per identificare nuovi geni attraverso il sequenziamentodell’intero esoma. Abbiamo sequenziato l’intero esoma di 4 membri della famiglia conLGMD1F separati da più di undici meiosi ed è stata identificata una singola condivisanuova variante eterozigote frame shift. Questo causa un'alterazione nonstop nel genedella Trasportina 3 (TNPO3) che codifica un membro della superfamiglia delle importineb. Per raggiungere il secondo compito, abbiamo prima reclutato 160 casi familiari didistrofia muscolare dei cingoli non specifica con un'apparente eredità autosomica. Tutti icampioni di DNA sono stati prima arricchiti con 486,480 bp, con una copertura di 2447esoni di 98 geni usando la tecnologia Haloplex con l’utilizzo di codici a barre. Abbiamoeseguito aggregati NGS di tutti i campioni e identificato un numero di mutazioni,verificate poi con sequenziamento Sanger. I casi sono inoltre stati studiati conpiattaforma AgilentMotorChip CGH array versione 3.0 per identificare delezioni oduplicazioni. Infine, nei casi selezionati, abbiamo eseguito RNA-Seq partendo da uncampione di biopsia muscolare. Abbiamo convertito mRNA in cDNA e lo abbiamopurificato con un personalizzato SureSelec t Target Enrichment System, focalizzatosugli stessi 98 mRNAs . Le sonde hanno una copertura 4x con un target totale di 1.41Mb di sequenze/campioni. Questi cDNA sono stati sequenziati usando codici a barrecercando di ottenere una copertura media di sequenziamento di 100x. I nostri risultaticonfermano che c’è un'eterogeneità molto alta nelle distrofie muscolari e che i test diDNA e RNA basati su NGS sono pronti per uso diagnostico.

Giugno 2012

Nuovi indici quantitativi di MRI utili per studiare malattie muscolari

C. Angelini, M. Fanin, E. Peterle (Padova, IT)

CONTESTO: Proponiamo nuovi modelli di misurazione quantitativa dell’atrofiamuscolare: l’indice del quadricipite (QI) e l’indice del vastus lateralis sinistro (VLI)misurando la loro area tramite MRI.METODO: Abbiamo usato sequenze T1 della MRI del muscolo della coscia, a circa 15cm dalla testa del femore (seconda slide della MRI nelle estremità inferiori). In questesequenze abbiamo misurato l’area muscolare del quadricipite femorale sinistro e delvastus lateralis sinistro. Queste misurazioni sono state compiute su 11 pazienti condiversi tipi di miopatie p.e. due casi di miopatie da accumulo di lipidi, una sclerosilaterale amiotrofica, 1 distrofia facio scapolo omerale, 1 miopatia miofibrillare, 1miopatia metabolica, 2 pazienti con LGMD2A, 1 paziente con LGMD1F, 1 miositeossificante, 1 miopatia aspecifica. Le biopsie muscolari di questi pazienti sono stateulteriormente analizzate con morfometria e marcatori molecolari dell’atrofia o autofagia,p.e. MURF, LC3.RISULTATI: Abbiamo eseguito la misurazione dell’area muscolare del quadricipitefemorale (QI) in 11 pazienti, che è risultata in media 3711 mm2 ± SD 792. In questogruppo di pazienti abbiamo identificato 2 sottogruppi, uno che include 5 pazienti con unalto grado di atrofia muscolare (gruppo con alta atrofia) i cui valori sono compresi tra2400 e 3400 mm2 (media 2966) e uno che include 6 pazienti con un basso grado diatrofia (gruppo con bassa atrofia), i cui valori sono compresi tra 3700 e 5000 mm2(media 4332).La misurazione dell’area muscolare del vastus lateralis in 11 pazienti era in media di963 mm2 ± 303. Nel sottogruppo atrofico il valore era compreso tra 400 e 900 mm2(media 658.7), mentre nel sottogruppo normale il valore era compreso tra 900 e 1400mm2 (media 1217.8)CONCLUSIONI: Sia l’indice dei quadricipiti che del vastus lateralis sembra utile pervalutare l’atrofia muscolare nelle LGMD, SLA e miopatie metaboliche: un alto grado diatrofia del QI è stato trovato nelle calpainopatie, malattie del motoneurone e distrofiamuscolare dei cingoli tipo 1F, la misurazione del VLM è apparsa meno specifica datoche comprende un'area più vasta. Entrambi questi indici quantitativi ottenuti dalla MRImuscolare, possono essere usati come risultati clinici della terapia in malattie


neuromuscolari in modo da seguire e studiare la storia naturale o gli effetti dei vari tipi diterapie (steroidi, carnitina, ecc.). Un promettente campo di ricerca appare essere lacorrelazione degli indici delle immagini con altri parametri di atrofia ottenuti nelle biopsiemuscolari, p.e. con sezione o fibre o marcatori molecolari dell’atrofia e autofagia.

Ottobre 2011

Atti della XI Conferenza dell’Associazione Italiana di MiologiaCagliari, Maggio 2011

LGMD1F – Un’ipotesi patogenetica basata sull’Istopatologia e ultrastruttura

G. Cenacchi, E. Peterle, L. Tarantino, V. Papa, M. Fanin, C. Angelini

Dipartimento clinico delle Scienze Radiologiche e Istopatologiche, Università di Bologna Dipartimento diNeuroscienze e VIMMM, Università di Padova

In una numerosa famiglia italospagnola con apparente eredità autosomica dominante èstata segnalata debolezza muscolare dei muscoli prossimali degli arti e dei muscoliassiali. Sono state descritte le caratteristiche cliniche, genetiche e istologiche in 5/32pazienti. È stato precedentemente identificato il locus nel cromosoma 7q32.1-32.2, manessun difetto è stato rilevato nella Filamina C, un gene candidato da questa regionecromosomica che codifica proteine leganti l’actina altamente espresse nel muscolo.Resocontiamo uno studio clinico-patologico di due pazienti (madre e figlia) della stessafamiglia spagnola. L’età di esordio è stato nell’adolescenza: un esordio più precocenella figlia con una debolezza più veloce conferma un’apparente anticipazione genetica.I risultati morfologici sono stati simili in entrambi i casi: H&E rileva un’aumentatavariabilità della dimensione delle fibre, atrofia delle fibre, tessuto connettivo endomisio eperimisio e vacuoli positivi alla fosfatasi acida. Lo studio ultrastrutturale conferma atrofiadelle fibre, anormali aggregati mitocondriali e vacuoli autofagosomali contenenti detriticellulari e immagini di pseudo mielina: non sono state riscontrate inclusioni filamentoseche sono solitamente associate a HIBM (miopatia ereditaria da corpi inclusi). Moltealterazioni di componenti miofibrillari sono state facilmente rilevate così come unimportante disordine, strutture bastoncellari con aspetto granulare e occasionalmentecorpi citoplasmatici. I nostri dati morfologici sostengono l’ipotesi che altre proteinecodificanti actina come FSCN3 e KIAA0265 della stessa regione critica, possanorappresentare degli interessanti geni candidati nel meccanismo patogenetico nellaLGMD1F.

Agosto 2003

Una nuova distrofia muscolare dei cingoli autosomica dominante (LGMD1F)mappata nel 7q32.1–32.2

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

1. Centre d’Investigacions en Bioquímica i Biologia Molecular (CIBBIM) Ospedale Universitario Valld’Hebron, Barcellona, Spagna;

2. Servei de Neurologia, Ospedale Universitario Vall d’Hebron, Barcellona, Spagna;3. Dipartimento di Neurologia, Columbia University College of Physicians and Surgeons, New York,

USA;4. Dipartimento di Biologia Cellualare, University of Potsdam, Germania;5. Dipartimento di Neurologia, University of Medicine and Dentistry New Jersey Medical School,

Newark, NJ.


RIASSUNTO: Nel 2001, gli autori descrivono le caratteristiche cliniche di una distrofiamuscolare dei cingoli autosomica dominante (LGMD1F) geneticamente distinta.Esaminando l’intero genoma con più di 400 marcatori microsatelliti, gli autori hannoidentificato una nuova malattia LGMD il cui locus è nel cromosoma 7q32.1-32.2.All’interno di questa regione cromosomica, Filamina C, un gene che codifica proteineleganti l’actina altamente espresse nel muscolo, era un ovvio gene candidato, tuttaviagli autori non hanno rilevato nessun difetto nella Filamina C o il suo prodotto proteico.

Le distrofie muscolari dei cingoli (LGMD) comprendono un gruppo eterogeneo dimalattie ereditarie caratterizzate da una progressiva e predominante debolezza deimuscoli prossimali con segni istologici di necrosi e rigenerazione nel muscolo. Ad oggi,15 forme geneticamente diverse di LGMD sono state identificate.Due anni fa, abbiamo descritto le caratteristiche cliniche, istologiche e genetiche diun’ampia famiglia spagnola di oltre 5 generazioni con LGMD e apparente ereditàautosomica dominante (AD); 44 di 76 (58%) figli di genitori malati manifestano lamalattia. L’esame clinico di 61 persone ha mostrato una debolezza muscolareprogressiva in 32, colpendo principalmente i muscoli dei cingoli pelvico e scapolare.L’analisi del linkage genetico molecolare per esaminare i loci cromosomici associati adaltre forme di LGMD autosomiche dominanti hanno dimostrato che questa parentela hauna diversa forma genetica di LGMD–AD. Per localizzare il locus cromosomico dellamalattia, abbiamo intrapreso uno scan dell’intero genoma usando marcatorimicrosatellite.

Agosto 2000

Distrofia muscolare dei cingoli autosomica dominante. Un’ampia parentela consegni di anticipazione

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

1. Dipartimento di Neurologia, Ospedale Universitario Vall d’Hebron, Barcellona, Spagna;2. Centre d’Investigacions en Bioquímica i Biologia Molecular (CIBBIM) Ospedale Universitario Vall

d’Hebron, Barcellona, Spagna;3. Dipartimento di Patologia e Neuropatologia, Ospedale di Meixoeiro;4. Dipartimento di Neurofisiologia Clinica, Ospedale Xeral-Cies, Vigo, Spagna;5. H. Houston Merritt Clinical Research Center for Muscular Dystrophy and Related Diseases,

Dipartimento di Neurologia, Columbia University College of Physicians and Surgeons, New York;

RIASSUNTO: 14 forme di distrofia muscolare dei cingoli (LGMD) geneticamente diversesono state identificate, inclusi 5 tipi autosomiche dominanti (LGMD-AD).OBIETTIVO: Descrivere le caratteristiche cliniche, istologiche e genetiche di una vastafamiglia con LGMD e apparente eredità autosomica dominante di oltre 5 generazioni. METODO: gli autori hanno esaminato 61 membri della famiglia; sono state eseguitebiopsie muscolari a 5 pazienti. L’analisi del linkage ha valutato i loci cromosomiciassociati ad altre forme di LGMD-AD.RISULTATI: Un totale di 32 individui hanno debolezza dei cingoli scapolare e pelvico. Laseverità sembra peggiorare nelle successive generazioni. I risultati della biopsiamuscolare sono stati non specifici e compatibili con distrofia muscolare. L’analisi dellinkage con i cromosomi 5q31, 1q11-q21, 3p25, 6q23, e 7q, ha dimostrato che questamalattia non è allelica alle LGMD tipo 1A, 1B, 1C, 1D e 1E.CONCLUSIONI: questa famiglia ha una forma geneticamente diversa di LGMD-AD. Gliautori stanno al momento eseguendo uno scan dell’intero genoma per identificare illocus della malattia.

21


Pubblicazioni scientifiche


LETTER TO THE EDITOR

INCOMPLETE PENETRANCE INLIMB-GIRDLE MUSCULARDYSTROPHY TYPE 1F

Limb-girdle muscular dystrophy (LGMD) type 1F (MIM #608423) is a rare autosomal dominant disorder whoselocus was mapped1,2 and gene identified3,4 by investigatingthe same large family. During examination of this family,we expanded the original pedigree and the clinical fea-tures of the disease. It is characterized by a variable degreeof muscle weakness and functional impairment, with onsetof symptoms either before age 15 (juvenile form) or inthe third to fourth decades (adult form).5 The clini-cal2genetic investigation of this family revealed that, insome patients, the disease was transmitted through appa-rently unaffected parents (incomplete penetrance). Tocalculate the exact penetrance rate, we examined both theclinical phenotype and the genotype of 115 family mem-bers. The attribution of clinical status (either affected orunaffected) was based on a neuromuscular examinationperformed by the same physician using a standardizedprotocol and by a questionnaire that we designed to iden-tify main disease symptoms (i.e., muscle weakness, gait dif-ficulty, and dysphagia). One hundred fifteen individuals(including the 27 subjects investigated in the originalsearch for the gene defect in this family3 and 88 newindividuals previously untested) underwent DNA samplecollection (obtained after written consent). The genotype(either mutant or non-mutant) was defined using amutation-specific test [amplification refractory mutationsystem2polymerase chain reaction (ARMS-PCR)] and con-firmed by DNA sequencing. The mutation segregating inthis family (c.2771delA in TPNO3 gene encoding transpor-tin-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 unaf-fected. Two unaffected subjects were younger than age 15years, with a future chance of developing the disease, and4 were adult “non-penetrant” individuals (at a mean age of31.5 years). Furthermore, 3 additional unaffected adultswhose DNA was unavailable, were obligate carriers ofthe disease based on the pattern of inheritance. Overall,the penetrance rate was estimated to be 84.7%.

We observed that clinical signs and symptoms of thedisease were progressively more likely to manifest withincreasing 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 individu-

als (numbers in parentheses indicate number of individuals in

each age group).VC 2014 Wiley Periodicals, Inc.

Letter to the Editor MUSCLE & NERVE Month 2015 1

December 9th, 2014


the penetrance is age-dependent. Incomplete penetrancemay be the effect of modifier genes or may due toenvironmental factors. Although the contribution ofepigenetic factors was not explored in this study, we didinvestigate the potential role of lifestyle and associatedconditions in determining disease manifestations.

These data show that age-related penetrance is acharacteristic feature of LGMD1F that reduces the pre-dictive value of the genetic test. Because age-related pen-etrance is a major challenge when attempting toquantify the genetic risk of a patient’s offspring, ourresults may be useful for genetic counseling, especiallyin 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

Vincenzo Nigro, MD, PhD3,4

Corrado Angelini, MD1,2

1Department of Neurosciences, University of Padova, Padova,Italy

2IRCCS Fondazione San Camillo Hospital, Venice, Italy

3Department of Biochemistry, Biophysics and GeneralPathology, II University of Naples, Naples, Italy

4Telethon 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 largekindred with evidence for anticipation. Neurology 2001;56:450–454.

2. Palenzuela L, Andreu AL, Gamez J, G�amez J, Vil�a 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 3as the causative gene for LGMD1F. PLoS One 2013;8:1–7.

4. Meli�a MJ, Kubota A, Ortolano S, V�ılchez JJ, G�amez J, Tanji K, et al.Limb-girdle muscular dystrophy 1F is caused by a microdeletion inthe 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 pathol-ogy of LGMD1F. J Neurol 2013;260:2033–2041.

Published online 00 Month 2014 in Wiley Online Library(wileyonlinelibrary.com). DOI 10.1002/mus.24539

---------------------------------------------------------

2 Letter to the Editor MUSCLE & NERVE Month 2015

December 9th, 2014


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

Acta Myologica • 2014; XXXIII: p. 1-12

1

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]

Limb-girdle muscular dystrophies (LGMD) are a highly het-erogeneous 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-X-linked, 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 pheno-types. 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 gen-eration 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 ba-sis 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 domi-nant and 23 autosomal recessive. The dominant forms (LGMD1) are: LGMD1A (myotilin), LGMD1B (lamin A/C), LGMD1C (ca-veolin 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 sarco-glycan), LGMD2G (telethonin), LGMD2H (TRIM32), LGMD2I(FKRP), LGMD2J (titin), LGMD2K (POMT1), LGMD2L (anoc-tamin 5), LGMD2M (fukutin), LGMD2N (POMT2), LGMD2O(POMTnG1), LGMD2P (dystroglycan), LGMD2Q (plectin), LG-MD2R (desmin), LGMD2S (TRAPPC11), LGMD2T (GMPPB),LGMD2U (ISPD), LGMD2V (Glucosidase, alpha ), LGMD2W(PINCH2).

Key words: Limb-girdle muscular dystrophies, LGMD, NGS

IntroductionThe term limb-girdle muscular dystrophy refers to a

long list of Mendelian disorders characterized by a pro-gressive deterioration of proximal limb muscles. Very of-ten, other muscles are affected, together with the heart

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 al-lowing affected people to have fairly normal life spans and activity levels (1). The term LGMD is becoming de-scriptive 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 child-hood onset also result in dramatic physical weakness and a shortened life-span. The advent of next generation se-quencing approaches has accelerated the pace of discov-ery 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 autoso-mal recessive.

Autosomal dominant LGMdThe LGMD1, i.e. the autosomal dominant forms,

have usually an adult-onset and are milder, because affect-ed 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).

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-disk-associated 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 my-ofibrillar myopathy. LGMD1A is characterized by late

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Table 1. Autosomal dominant limb girdle muscular dystrophy.

Gene Clinical phenotype

Disease Locus Name Exons Protein (protein function)

Typical onset Progression Cardiomiopathy sCK Allelic disorders (OMIM, #)

LGMD1A 5q31.2 TTID 10 myotilin (structural; Z disc) Adulthood Slow Not observed 3-4X

Myopathy, myofibrillar, 3 (609200)Myopathy, spheroid body (182920)

LGMD1B 1q22 LMNA 12lamin A/C(structural; fibrous nuclear lamina )

Variable (4-38y) Slow Frequent 1-6X

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)Heart-hand syndrome, Slovenian type(610140)Hutchinson-Gilford progeria(176670)Lipodystrophy, familial partial, 2(151660)

Malouf syndrome(212112)

Mandibuloacral dysplasia(248370)Muscular dystrophy, congenital(613205)Restrictive dermopathy, lethal(275210)

LGMD1C 3p25.3 CAV3 2

caveolin 3(scaffolding protein within caveolar membranes)

Childhood Slow/moderate Frequent 10X

Cardiomyopathy, familial hypertrophic(192600)Creatine phosphokinase, elevated serum(123320)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 -

LGMD1E 2q35 DES 9desmin (structural; intermediate filament)

Adulthood Slow Frequent 5-10X

Muscular dystrophy, limb-girdle, type 2R(615325)Cardiomyopathy, dilated, 1I(604765)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 -

LGMD1G 4q21 HNRPDL 9

Heterogeneous nuclear ribonucleoprotein D-like protein (ribonucleoprotein, RNA-processing pathways)

Variable (13-53y) Slow Not observed 1-9X -

LGMD1H 3p23-p25 - - - Variable (10-50y) Slow Not observed 1-10X -

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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 ex-ons and encodes DnaJ homolog, subfamily B, member 6. DNAJ family members are characterized by a highlyconserved amino acid stretch (2) called the ‘J-domain’.They exemplify a molecular chaperone functioning ina wide range of cellular events, such as protein foldingand oligomeric protein complex assembly (11). Mis-sense 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 clear-ance of misfolded proteins. Functional testing in vivohave shown that the mutations have a dominant toxic ef-fect mediated specifically by the cytoplasmic isoform ofDNAJB6. In vitro studies have demonstrated that the mu-tations increase the half-life of DNAJB6, extending thiseffect to the wild-type protein, and reduce its protectiveanti-aggregation effect.

DNAJB6 is located in the Z line and interacts with BAG3. Mutations in BAG3 are known to cause myofibril-lar 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 myo-pathy.

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 sar-torius and the anterolateral lower leg muscles are mostly spared. DNAJB6 gene mutations may also be associated with distal-predominant myopathy. Symptoms in the up-per limbs appear later. Some patients develop calf hyper-trophy. Onset ranges from 25 to 50 years, with some pa-tients 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.

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 cor-rectly, as a form of autosomal dominant desminopathy or myofibrillar myopathy. This form is also known as dilated cardiomyopathy type 1F (CMD1F). One family previous-ly categorized as having LGMD and dilated cardiomyo-pathy was reported, indeed, to have the splice site muta-tion IVS3+3A>G in the desmin (DES) gene at 2q35 (14).

onset proximal weakness with a subsequent distal weak-ness. Some patients show nasal and dysarthric speech. Serum CK is normal or mildly elevated. Muscle pathol-ogy shows rimmed vacuoles with or without inclusions. Electron microscopy shows prominent Z-line streaming. Cardiac and respiratory involvement occasionally occurs.

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 splic-ing 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, rang-ing from mechanical nuclear membrane maintenance to gene regulation. The ‘laminopathies’ comprise different well-characterized phenotypes, some of which are con-fined to the skeletal muscles or skin, while others are multi-systemic, such as lipodystrophy, Charcot-Marie Tooth disease, progeroid syndromes, dilated cardiomyo-pathy and Emery-Dreifuss muscular dystrophy (EDMD). The LGMD1B is characterized by a symmetric proximal weakness starting from the legs, associated with atrioven-tricular 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 LG-MD1B and EDMD (8). Usually the more severe forms of EDMD with a childhood onset have missense mutations, whereas the milder LGMD1B is associated with het-erozygous 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 en-codes a 1.4kb mRNA composed of only two exons. Caveo-lin-3 is a muscle-specific membrane protein and the prin-cipal 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.

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-

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Autosomal recessive LGMDThe 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 pheno-type (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 disor-der. 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).

LGMD2A - LGMD2A is caused by Calpain 3 (CAPN3) gene mutations and represents the most fre-quent 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 heterozy-gotes (1:100), carrying many different CAPN3 patho-genic changes. Calpains are intracellular nonlysosomal cysteine proteases modulated by calcium ions. A typical calpain is a heterodimer composed of two distinct subu-nits, 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-cal-pain and micro-calpain), but contains specific insertion sequences (NS, IS1 and IS2). Calpains cleave target pro-teins 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).

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 pro-tein involved in calcium-mediated sarcolemma resealing. LGMD2B is the second most frequent LGMD2 form (15-25%) in numerous countries, but not everywhere (23). Muscle inflammation is recognized in dysferlinopathy and dysferlin is expressed in the immune cells.

For desmin see also LGMD2R. As in the desminopathies, LGMD1E family members show dilated cardiomyopathy and conduction defects together with progressive proxi-mal muscle weakness starting in the second or third dec-ade. 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 subsar-colemmal 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).

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 iden-tification 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% pen-etrance. The TNPO3 gene is composed of 23 exons and encodes a 923-amino acid protein, also expressed in skel-etal 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 lat-eralis and the ileopsoas muscle (16). Interestingly, some patients present with dysphagia, arachnodactyly and res-piratory insufficiency. CK range is 1-3x. No cardiac in-volvement has been reported.

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 pheno-type. Patients show late-onset LGMD associated with pro-gressive fingers and toes flexion limitation (18).

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 fourth-fifth decade of life.

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Table 2. Autosomal recessive limb girdle muscular dystrophy.


Disease Locus Name Exons Protein product LGMD phenotype Typical onset Progression Cardiomiopathy sCK Allelic disorders

(OMIM, #)

LGMD2A 15q15 CAPN3 24 Calpain 3 ordinary Adolescence Moderate/rapid Rarely observed 3–20X

LGMD2B 2p13.2 DYSF 56 Dysferlin ordinary Young adulthood Slow Possible 5-40X

Miyoshi muscular dystrophy 1 (254130)Myopathy, distal, with anterior tibial onset (606768)

LGMD2C 13q12 SGCG 8 γ-Sarcoglycan ordinary Early childhood Rapid Often severe 10–70X

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

LGMD2J 2q24.3 TTN 312 or more Titin occasional Young

adulthood Severe Not observed 10-40X

Cardiomyopathy, dilated, 1G (604145)Cardiomyopathy, familial hypertrophic, 9 (613765)Myopathy, early-onset, with fatal cardiomyopathy (611705)Myopathy, proximal, with early respiratory muscle involvement (603689)Tibial muscular dystrophy, tardive (600334)

LGMD2K 9q34.1 POMT1 20 Protein-O-mannosyltransferase 1 occasional Childhood Slow Not observed 10-40X

Muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 1 (236670)Muscular dystrophy-dystroglycanopathy (congenital with mental retardation), type B, 1 (613155)Muscular dystrophy-dystroglycanopathy (limb-girdle), type C, 1 (609308)

LGMD2L 11p13-p12 ANO5 22 Anoctamin 5 ordinaryVariable (young

to late adulthood)

Slow Not observed 1-15X

Gnathodiaphyseal dysplasia (166260)Miyoshi muscular dystrophy 3 (613319)

LGMD2M 9q31 FKTN 11 Fukutin occasional Early childhood Moderate Possible 10-70X

Cardiomyopathy, dilated, 1X (611615)Muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 4 (253800)Muscular dystrophy-dystroglycanopathy (congenital without mental retardation), type B, 4 (613152)

(continues)

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Disease Locus Name Exons Protein product LGMD phenotype Typical onset Progression Cardiomiopathy sCK Allelic disorders

(OMIM, #)

LGMD2N 14q24 POMT2 21 Protein-O-mannosyltransferase 2 occasional Early childhood Slow Rarely observed 5-15X

Muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 2 (613150)Muscular dystrophy-dystroglycanopathy (congenital with mental retardation), type B, 2 (613156)

LGMD2O 1p34.1 POMGnT1 22

Protein O-linked mannose beta1,2-N-acetylglucosaminyl

transferase

occasional Late childhood Moderate Not observed 2-10X

Muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 3 (253280)Muscular dystrophy-dystroglycanopathy (congenital with mental retardation), type B, 3 (613151)Muscular dystrophy-dystroglycanopathy (limb-girdle), type C, 3 (613157)

LGMD2P 3p21 DAG1 3 Dystroglycan singular Early childhood Moderate Not observed 20X

LGMD2Q 8q24 PLEC1 32 Plectin singular Early childhood Slow Not observed 10-50X

Epidermolysis bullosa simplex with pyloric atresia (612138)Epidermolysis bullosa simplex, Ogna type (131950)Muscular dystrophy with epidermolysis bullosa simplex (226670)

LGMD2R 2q35 DES 9 Desmin (structural; intermediate filament) occasional Young

adulthoodA-V conduction

block 1X

Muscular dystrophy, limb-girdle, type 2R(615325)Cardiomyopathy, dilated, 1I(604765)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

LGMD2T 3p21 GMPPB 8 GDP-mannose pyrophosphorylase B occasional

Early childhood-

Young adulthood

Possible

Muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 14 (615350)Muscular dystrophy-dystroglycanopathy (congenital with mental retardation), type B, 14 (615351)

LGMD2U 7p21 ISPD 10 Isoprenoid synthase domain containing occasional Early / Late Rapid/

Moderate Possible 6-50X

Muscular dystrophy-dystroglycanopathy (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 -

Table 2. (follows).

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The “dysferlinopathies” include limb-girdle muscu-lar dystrophy type 2B (LGMD2B) and the allelic forms Miyoshi myopathy (MM), which is an adult-onset dis-tal form, and distal myopathy with anterior tibialis on-set (DMAT), but varied phenotypes are observed. LG-MD2B affects earlier the proximal muscles of the arms whereas MM affects the posterior muscles of the leg.

DYSF gene mutations are associated with heteroge-neous clinical pictures ranging from severe functional disability to mild late-onset forms (24). About 25% of cases are clinically misdiagnosed as having polymyosi-tis (25). This classification into separate phenotypes does not reveal true disease differences (26) and the allelic forms are not due to different mutations. Additional fac-tors (e.g., additional mutations in neuromuscular disease genes or sport activities that include maximal eccentric contractions) may worsen the disease expression of caus-ative 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-FLoss-of-function mutations in any of the genes en-

coding the four members of the skeletal muscle sarcogly-can 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 re-gion and a large extra-cellular domain containing a clus-ter 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

named adhalin and contains a “dystroglycan-type” cad-herin-like domain that is present in metazoan dystrogly-cans (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 Z-disc proteins. Patients show adolescence-onset weakness initially affecting the proximal pelvic muscles and then the distal legs with calf hypertrophy. A homozygous non-sense 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 susceptibil-ity 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 com-posed 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, inter-actions 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) mu-tation (W4R) associated with dilated cardiomyopa-thy (DCM) results in a marked defect in Telethonin inter-action/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 compris-ing at least 70 human members sharing the tripartite mo-tif (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).

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LGMD2H is usually a late-onset condition charac-terized by proximal weakness, atrophy, and moderately raised levels of creatine kinase. Until 2008, the only LG-MD2H mutation was Asp487Asn found in Hutterite fam-ilies (40). Different TRIM32 mutations were then identi-fied 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 associ-ated with STM morphotype (42).

LGMD2I, LGMD2K, LGMD2M, LGMD2N, LGMD2O, and LGMD2P

The name dystroglycanopathy has been given to de-fects due to mutations in six genes (POMT1, POMT2, POMGnT1, FKTN, FKRP and DAG1) (43). These varia-tions reduce dystroglycan glycosylation and cause a wide range of phenotypes ranging from mild congenital mus-cular 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 congen-ital muscular dystrophies, as well as in LGMD2I. Fuku-yama-type congenital muscular dystrophy (FCMD), is one of the most common autosomal recessive disorders in Japan characterized by a congenital muscular dystro-phy 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 in-volved in LGMD2I (15).

All of these diseases are associated with changes in alpha-dystroglycan expression due to a glycosylation de-fect of alpha-dystroglycan. Dystroglycan is normally ex-pressed and recognized by polyclonal antibodies, but it is abnormally glycosylated and not recognized by monoclo-nal 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 com-mon cause of LGMD, accounting for at least 10% of all LGMD with either severe or mild phenotypes (47, 48).

LGMD2J - TTN is one of the most complex human genes. The titin gene spans 294,442 bp of genomic se-quence 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 mu-tation in the C terminus of titin (FINmaj 11bp deletion/insertion) causes LGMD2J (50). Titin is the giant sarco-meric 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 muscu-lar 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 second-ary 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 polymor-phisms may have a confounding effect on the diagnosis.

LGMD2K - LGMD2K is caused by hypomorphic missense mutations in the POMT1 gene at 9q34, contain-ing 20 exons and spanning about 20 kb. Mutations allow-ing a residual enzyme activity are linked to mild forms. Different POMT1 alleles, cause congenital muscular dystrophies due to defects of the dystroglycan glycosyla-tion (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 cod-ing 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 North-ern Europe encompassing 10%-20% of cases (61). The penetrance is probably incomplete, since females are less frequently affected than males. The most common muta-tion in Northern Europe is c.191 dupA in exon 5 (62). Pa-tients are usually ambulant and the onset is in adulthood. They show asymmetric muscle involvement with preva-lent quadriceps atrophy and pain following exercise. CK levels are 5-20x. There is no evidence for contractures,

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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 ac-ids. 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 congeni-tal 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 in-volvement that deteriorates with acute febrile illness. Im-provement 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, contain-ing 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 char-acterized 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 muta-tions 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 dupli-cation (72).

LGMD2P - LGMD2P is caused by specific changes of the dystroglycan (DAG1) gene itself. Recently, Camp-bell 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-specif-ic 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-

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 notshow skin pathology. LGMD2Q patients show early-on-set 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. Au-tosomal dominant mutations in the DES gene are associ-ated 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 af-fecting both the upper and lower limbs. The serum Ck is normal. LGMD2R patients usually show A-V con-duction blocks but no cardiomyopathy.

LGMD2S - This is caused by mutation in the trans-port 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 identi-fied in a consanguineous Syrian family with an uncharac-terized form of LGMD and in five Hutterite individuals presenting with myopathy, ID, hyperkinetic movements and ataxia (78).

TRAPPC11 is a transport protein particle compo-nent involved in anterograde membrane transport from the endoplasmic reticulum (ER) to the ER-to-Golgi in-termediate compartment (ERGIC) in mammals (79). Mu-tations identified so far (c.2938G>A/ p.Gly980Arg and c.1287+5G>A) cause modifications in TRAPP complexcomposition, in Golgi morphology and in cell traffick-ing. The LGMD2S pathogenic mechanism is similar tothat causing Danon disease, an X-linked myopathy dueto LAMP2 mutations and affecting the secretory path-way (80).

The LGMD2S phenotype ranges from a slowly pro-gressive LGMD with childhood onset and high CK to a syndrome characterized by myopathy but also neurologi-cal 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-

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so developed decreased ejection fraction with global left ventricular dysfunction in their 3rd decade, severe quadri-paresis and relative sparing of the face, and characteristi-cally 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 com-plex. We tried to reorganize the different genes so far de-scribed following the traditional nomenclature. However for the autosomal recessive forms there are few letters available. The next forms will be LGMD2X, LGMD2Y and LGMD2Z. We propose, after the LGMD2Z form, the acronyms LGMD2AA, LGMD2AB, LGMD2AC, etc. to avoid renaming consolidated definitions thereby generat-ing even higher confusion.

AcknowledgementsThis study was mainly supported by grants from

Telethon, Italy (TGM11Z06 to V.N.) and Telethon-UILDM (Unione Italiana Lotta alla Distrofia Musco-lare) (GUP 10006 and GUP11006 to V.N.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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LGMD2U - This is the form caused by some par-ticular alleles of the isoprenoid synthase domain contain-ing (ISPD) gene. The ISPD gene spans 333kb at chro-mosome 7p21 and contains 10 exons. ISPD mutations disrupt dystroglycan mannosylation and cause of Walker-Warburg syndrome (82, 83). Mutations in ISPD as well as TMEM5 genes have been associated with severe cob-blestone 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 muta-tions in the ISPD gene and LGMD was, however, older than that of forms 2P-2T, but to avoid discordant defini-tions 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 muta-tions 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 mus-cle 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-

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68. Biancheri R, Falace A, Tessa A, et al. POMT2 gene mutation in

May 2014


Ho pital Cochin, Paris,France; 7 AP-HP, Service d’imagerie medicale, Ho pital Raymond Poincare, Garches, Garches, France

To determine the clinical characteristics of limb-girdle muscular dys-trophy 2E (LGMD2E), and to analyze the genetic and histopathological features. All LGMD2E patients followed at three European neuromuscu-

lar centres were included. The past medical history was collected, and dis-ease course was evaluated by specific questionnaires. Molecular analysis of SGCB gene and histopathological features were reviewed. 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 pop-ulations 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 21Human Genome Research Center, Institute of Biosciences, University of

Sa o Paulo, Genetics and Evolutionary Biology, Sa o Paulo, Brazil; 2 Uni-versity of Sa o Paulo, Genetics and Evolutionary Biology, Sa 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. Out-

side 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 pre-dominant European genetic background, we consider that the common mutation arose in Europe and spread through Brazilian population. How-

ever, 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: FAPESP-

CEPID, 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 11Institute 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 anocta-

minopathy. 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 com-

mon 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

1University of Padova, Padova, Italy; 2University of Bologna, Bologna,

Italy; 3TIGEM, 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 his-

topathology 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 hyp-

otrophy both in proximal upper and in lower extremities in calf muscles.

The severity was not increased in successive generations. Unreported clin-

ical 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

766 Abstracts / Neuromuscular Disorders 23 (2013) 738–852

October, 2013


of progressive atrophy and myofiber loss. Electron microscopy revealed

accumulation of myofibrillar bodies in muscle fibers. Accumulation of des-

min 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 pheno-

type 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.

http://dx.doi:10.1016/j.nmd.2013.06.462

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 11Washington University, Neurology, St Louis, United States; 2 Washington

University, Cell Biology and Physiology, St Louis, United States; 3 Cedars-

Sinai 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 func-tion may underlie the molecular basis of many degenerative disorders including some myopathies. The pathogenic mechanism of these chaper-onopathies 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 pro-tein 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, homolo-

gous 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 cli-ent 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 inter-play 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

1Columbia University Medical Centre, Department of Neurology, New

York, United States; 2Vall dHebron Institut de Recerca, Universitat

Autonoma de Barcelona, Research Group on Neuromuscular and Mito-

chondrial Disorders, Barcelona, Spain; 3 Institute of Biomedical Research

of Vigo (IBIV), University Hospital of Vigo (CHUVI), Department of

Pathology and Neuropathology, Vigo, Spain; 4Hospital Universitari i

Politecnic La Fe, Department of Neurology, Valencia, Spain; 5Hospital

Universitari Vall dHebron, Institut de Recerca, Universitat Autonoma de

Barcelona, Neuromuscular Disorders Clinic,Department of Neurology,

Barcelona, Spain; 6Columbia University Medical Centre, Department of

Pathology and Cell Biology, New York, United States; 7Centro Nacional

de Analisis Genomico, Barcelona, Spain

Limb-girdle muscular dystrophy 1F (LGMD1F) is an autosomal dom-

inant muscular disease affecting a Spanish family. Using whole genome

sequencing, we identified a single nucleotide deletion (c.2771del) in trans-

portin-3 gene (TNPO3) in a LGMD1F patient. The mutation disrupts the

termination codon of TNPO3 and causes a reading frame shift. Transpor-

tin-3 is a nuclear protein, and mediates import of serine–arginine rich pro-

teins 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 ana-

lyzed 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. Hema-

toxylin-eosin (HE) stained muscle revealed nuclei (10.7 ± 3.0%;

mean ± SD) with central pallor in all patients studied. Immunohisto-

chemistry 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 stain-

ing 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.

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 1Neuromuscular Research Center, Tampere University Hospital, Neurol-

ogy, Tampere, Finland; 2 Neuromuscular Research Center, Tampere Uni-

versity Hospital, Pathology, Tampere, Finland

Limb girdle muscular dystrophies are a large group of both domi-

nantly 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 immu-

nohistochemistry and semithin sections with subsequent electron microscopy.

Uniform findings were myopathic/dystrophic changes in all patients. Restricted and easily overlooked myofibrillar pathology in routine histo-pathology included protein aggregates reactive for Z-disk proteins such

Abstracts / Neuromuscular Disorders 23 (2013) 738–852 767

October, 2013


Distrofia dei cingoli, Telethon scopre il gene responsabile della rarapatologia

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."

May 7th, 2013


Next-Generation Sequencing Identifies Transportin 3 asthe Causative Gene for LGMD1FAnnalaura 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, 2Dipartimento di Biochimica Biofisica e Patologia Generale, Seconda Universita degli Studi di Napoli,

Napoli, Italy, 3Dipartimento di Neuroscienze, Universita degli Studi di Padova, Padova, Italy, 4Cancer Research UK, London, United Kingdom, 5 Fondazione IRCCS Istituto

Neurologico C. Besta, Milano, Italy, 6Dipartimento di Medicina clinica e sperimentale, Universita 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 largefamily 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. Wesequenced the whole exome of four family members and identified a shared heterozygous frame-shift variant in theTransportin 3 (TNPO3) gene, encoding a member of the importin-b super-family. The TNPO3 gene is mapped within theLGMD1F critical interval and its 923-amino acid human gene product is also expressed in skeletal muscle. In addition, weidentified an isolated case of LGMD with a new missense mutation in the same gene. We localized the mutant TNPO3around the nucleus, but not inside. The involvement of gene related to the nuclear transport suggests a novel diseasemechanism 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 Genefor 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 permitsunrestricted 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 Lottaalla Distrofia Muscolare) (GUP 10006 and GUP11006 to V.N.). This work was also supported by grants from the Association Francaise contre les Myopathies (13859to 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.

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

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, immuno-

fluorescence 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 limb-

girdle 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

PLOS ONE | www.plosone.org 1 May 2013 | Volume 8 | Issue 5 | e63536

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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 StatementThis 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).

PatientsNineteen 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.

Exome sequencing and analysisEnrichment 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

(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).

Mutation DetectionWe 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).

Immunoblotting AnalysisFor TNPO3 immunoblotting, muscle samples were homoge-

nized 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 Tris-

HCl, 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 Anti-

TNPO3 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 mem-

branes were incubated with the secondary antibody, goat anti-

mouse 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.

TransfectionPlasmid 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

TNPO3 Is the LGMD1F Gene


May, 2013


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 penicil-

lin-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 anti-

rabbit and/or Cy3-conjugated anti-mouse antibodies.

Results

Exome analysisThe 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

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

Figure 1. LGMD1F family pedigree. Squares represent male; circles represent female; white figures symbolize normal individuals; black figuresindicate individuals with clinical muscular dystrophy. The original LGMD1F family has been extended from subject II,2 and now includes 64 LGMDpatients 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 patientsindicated by arrows (V-28, VI-36, VI-53, VII-5).doi:10.1371/journal.pone.0063536.g001

Table 1. Total and Shared Variants in Patients with LGMD1F.

Patient variant type V-28 VI-53 VII-5Shared by allSOLiD VI-36 Shared by all four

exonic/splicing 21,105 21,366 17,123 5,722 17,183 4,212

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



May, 2013


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

Figure 2. Sequence analyses of the TNPO3 mutations. a) Heterozygous delA mutation in Exon 22 of the TNPO3 gene in Proband VII-5. Alignedelectropherograms show mutated (top) and wild-type (bottom) sequences; b) Heterozygous. c.G2453A) in exon 21 of the TNPO3 gene; c) Pedigree ofthe isolated case.doi:10.1371/journal.pone.0063536.g002

Figure 3. Western blot analysis of skeletal muscle tissue withantibodies to TNPO3. Equal amounts of muscle proteins from aLGMD1F patient and a control were run in each lane (10 mg) on a 9%SDS-polyacrylamide gel and then blotted onto nitrocellulose mem-brane. In this experiment, we used a monoclonal antibody thatrecognizes a recombinant fragment (Human) near the N terminus ofTNPO3 at a 1:100 dilution. A double band is visible in the patient only.doi:10.1371/journal.pone.0063536.g003



May, 2013


characteristic LGMD phenotype. Muscular histopathological data

evidenced dystrophic features and, in addition, discrete mitochon-

drial alterations, with sporadic ragged-red fibers and cytochrome c

oxidase negative fibers. Mutations in the mitochondrial DNA were

excluded.

Figure 4. Indirect immunofluorescence analysis of the wt-hTNPO3 compared with delA p.X924C -hTNPO3. Following transienttransfections, 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 wereHA-tagged (red). a) An accumulation around the nucleus is usually observed using the mutant delA p.X924C -hTNPO3. b) The typical intranuclearstaining 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



May, 2013


Importance of the TNPO3 mutationTo 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.

Discussion

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 TNPO3disease-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

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 dominant-

negative mechanism. This should be possible by the use of

antisense morpholino oligos in Danio rerio (Zebrafish) where a singleand 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.

Supporting Information

Table S1 Exome sequencing data.

(DOC)

Table S2 Primers designed for TNPO3 amplification and Sanger

sequencing.

(DOC)

Table S3 Exons inadequately covered by NGS exome sequenc-

ing 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 (HL-

102923), the WHI Sequencing Project (HL-102924), the Broad GO

Sequencing Project (HL-102925), the Seattle GO Sequencing Project (HL-

102926) 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 MMRR 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.

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May, 2013


ORIGINAL COMMUNICATION

Clinical phenotype, muscle MRI and muscle pathologyof 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. Widen-

ing 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

areas, autophagosomes and accumulation of myofibrillar

and cytoskeletal proteins. The LGMD1F is characterized

by a selective involvement of limb muscles with respira-

tory 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 imag-

ing 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

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 Neurologıa, 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

123

J Neurol (2013) 260:2033–2041

DOI 10.1007/s00415-013-6931-1

30 April 2013


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 out-

patient examination, organized by a family association.

The clinical data were collected in 24 patients, including

seven previously non-investigated cases, by both a com-

plete clinical neuromuscular examination and a standard-

ized 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 = tip-

toe 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).

Muscle samples were snap-frozen in liquid nitrogen-

chilled 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 impor-

tance 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 immunohis-

tochemistry using a panel of antibodies against desmin

(MAB1698, Chemicon, Temecula, CA, USA; 1:50), myo-

tilin (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 myo-

fibrillar and membrane components. For this purpose, 8 lmthick 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 con-

jugated Ig (Caltag, Burlingame CA) diluted 1:100 and

incubated for 30 min. Sections were mounted with anti-

fading 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 anti-

body against MuRF-1 (MP3401, ECM Biosciences,

Versailles, KY, USA), diluted 1:500 in TTBS. After a

thorough washing, the immunoreactive bands were

2034 J Neurol (2013) 260:2033–2041

123

30 April 2013


visualized using anti-rabbit peroxidase-conjugated anti-

bodies and the chemioluminescent method (GE Health-

care, 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 spin-

echo 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 distribu-

tion 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 expres-

sed as mm2.

Results

Pattern of inheritance

We studied a large LGMD1F family of Italian-Spanish

origin. In our study, the family pedigree has been recon-

structed 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 phenom-

enon, which has previously been suggested [1], was not

confirmed in our series of patients.

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 proxi-

mal 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 arachnodac-

tyly (Fig. 1), pes cavus, and mild Achilles tendon retrac-

tion. 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

J Neurol (2013) 260:2033–2041 2035

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30 April 2013


Fig. 1 Pictures from five

different LGMD1F patients.

Note atrophy of upper girdle

muscles, especially deltoid and

triceps brachii (a–e), causingdifficulty 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)

2036 J Neurol (2013) 260:2033–2041

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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 pro-

gressive, resulting in three patients in early loss of ambu-

lation (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 under-

went 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.

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 late-

ralis 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 neuromus-

cular controls (3,843 and 991 mm2, respectively).

Muscle histopathology, morphometry,

immunohistochemistry, immunoblotting

Muscle biopsies obtained from the two patients investi-

gated, 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-chair-

bound before age 30 years,

grade = 8) are indicated in

thick line; patients showing a

slower course are indicated in

thin line

J Neurol (2013) 260:2033–2041 2037

123

30 April 2013


atrophic and hypertrophic fibers (increased coefficient size

variability, cross sectional area, atrophy factor, and

hypertrophy factor). Conversely, in case 1, the most rele-

vant change was a generalized fiber atrophy (diameter,

atrophy factor, coefficient size variability), which was

significantly increased in the second biopsy done at a dis-

tance 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 intracyto-

plasmic 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 histopa-

thological 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 rela-

tively benign in most adult patients. Only three patients

have lost ambulation before age 30 years. Muscle

Fig. 3 Muscle MRI T1

sequences from two different

patients at the level of the thigh

(a, c) and calf (b). Note fibro-

fatty replacement and atrophy of

vastus lateralis in the thigh

(a, c) and triceps surae in the

leg (b)

2038 J Neurol (2013) 260:2033–2041

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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) andacid 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

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)

J Neurol (2013) 260:2033–2041 2039

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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 lim-

itation 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 quadri-

ceps 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 pro-

gressive muscle atrophy, myopathic EMG, fibro-fatty

replacement of muscle on MRI, and active changes in

muscle biopsy. The cytoplasmic inclusions and myofibril-

lar 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

0

25

µ

75

50

100

0

250

500

750

1000

Fiber diameter Fiber cross sectional area

Fiber atrophy factor

Case 1 (12 years)

Case 1 (28 years)

Case 2 (54 years)

m m2

0

2000

4000

6000

8000

10000

12000

14000

un

its

A

B

0

250

500

750

1000

Fiber hypertrophy factor

un

its

Case1

Case 2

Cntr1

Cntr2

* ** * *

*

Coefficient fiber size variability

0

200

400

600

800

un

its

µ

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 Coomassie-

stained gel, MuRF-1 protein

quantity was normal in case 1

(98 % of control mean) and

highly increased in case 2

(250 % of control mean)

2040 J Neurol (2013) 260:2033–2041

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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 involve-

ment is frequently found.

The muscles from our patients showed protein aggre-

gates 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 path-

ways 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 mem-

bers who promoted meetings for neuromuscular examination and

blood sample collection. This work was supported by grants from the

Association Francaise contre les Myopathies (13859 to MF, 14999

and 16216 to CA) and the Telethon Italy (GTB12001 and GUP10006

to CA and GUP11006 to UN).

Conflicts of interest The authors declare that they have no conflict

of interest.

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1. Gamez J, Navarro C, Andreu AL et al (2001) Autosomal domi-

nant limb-girdle muscular dystrophy: a large kindred with evi-

dence for anticipation. Neurology 56:450–454

2. Palenzuela L, Andreu AL, Gamez J et al (2003) A novel auto-

somal 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

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girdle muscular dystrophies, hyaline body myopathies and myo-

tonic dystrophies. Radiol Med 115:585–599

5. Starling A, Kok F, Passos-Bueno MR, Vainzof M, Zatz M (2004)

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trophy (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

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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.

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8. Cenacchi G, Peterle E, Fanin M, Papa V, Salaroli R, Angelini C

(2013) Ultrastructural changes in LGMD1F. Neuropathology.

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BRAINA JOURNAL OF NEUROLOGY

Limb-girdle muscular dystrophy 1F is caused by amicrodeletion in the transportin 3 geneMaria J. Melia,1,2,* Akatsuki Kubota,3,* Saida Ortolano,4,* Juan J. Vılchez,5 Josep Gamez,6

Kurenai Tanji,7 Eduardo Bonilla,3,7,† Lluıs Palenzuela,1,2 Israel Fernandez-Cadenas,1

Anna Pristoupilova,8,9 Elena Garcıa-Arumı,1,2 Antoni L. Andreu,1,2 Carmen Navarro,2,4

Michio Hirano3,# and Ramon Martı1,2,#

1 Research Group on Neuromuscular and Mitochondrial Disorders, Vall d’Hebron Institut de Recerca, Universitat Autonoma 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 Politecnic La Fe, Valencia, 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 Autonoma 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 Analisis Genomico, 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 Martı, PhD,

Research Group on Neuromuscular and Mitochondrial Disorders,

Vall d’Hebron Institut de Recerca, VHIR, Universitat Autonoma 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]

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/arginine-

rich 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, expres-

sion of the mutant messenger RNA and histological abnormalities of nuclei and TNPO3 indicate altered TNPO3 function. Our

doi:10.1093/brain/awt074 Brain 2013: 136; 1508–1517 | 1508

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]

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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

IntroductionThe limb-girdle muscular dystrophies (LGMDs) comprise a group

of genetically heterogeneous disorders characterized by progres-

sive and predominantly proximal muscle weakness with histolo-

gical 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 categor-

ization of LGMD into autosomal dominant-LGMD (LGMD1) and

autosomal recessive (LGMD2) forms is being refined by a classifi-

cation 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 indi-

viduals 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 pre-

sented here.

Initially, the presence of rimmed vacuoles and filamentous

inclusions in myofibres of affected subjects prompted us to con-

sider a diagnosis of hereditary inclusion body myopathy (Huizing

and Krasnewich, 2009). Hereditary inclusion body myopathy typ-

ically presents as an autosomal recessive trait and is due to mu-

tations 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 7q22.1-

31.1 (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, contain-

ing 66 genes. These data confirmed that this family has a genet-

ically 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, to-

gether with the molecular results at DNA, RNA and protein

levels, fully support the pathogenic role of this mutation in

LGMD1F.

Materials and methods

PatientsThe 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, Valencia, Spain). Functional ac-

tivity 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, moth-

eaten appearance with scattered small areas of increased signal invol-

ving 530% of muscle volume; 3, moderate involvement (a late moth-

eaten 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 con-

fluent 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.

Muscle biopsiesMuscle 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, IV-

11 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

LGMD1F is caused by a TNPO3 mutation Brain 2013: 136; 1508–1517 | 1509

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CM100 transmission electron microscope equipped with a digital

camera and the ITEM SYSTEM VELETA (FEI Company) software.

DNA and RNA isolation andcomplementary DNA synthesisDNA 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, complemen-

tary DNA was synthesized using the High Capacity complementary

DNA Reverse Transcription kit, which uses random hexamers

(Applied Biosystems).

Whole genome sequencingSequencing 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

300–700bp. 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 adap-

ter-insert fragments (two insert sizes: 430 bp and 460bp) were ampli-

fied with 10 PCR cycles and sequenced on an Illumina HiSeq 2000

platform with paired end run of 2 � 100bp. 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-value5 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 sequencingDNA 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 indi-

viduals 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–restrictionfragment length polymorphism analysisA 629bp fragment was PCR-amplified (Forward primer

5’-TCTACTACCCTGGACCACCG-3’ and Reverse primer

5’-CACACCCCCAAACAGGAACT-3’) from skeletal muscle comple-

mentary 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 frag-

ments: 617 bp + 12 bp. The c.2771del mutation generates an add-

itional target, producing the expected restriction fragment pattern of

400 bp + 216bp + 12bp. The fragments were resolved by electro-

phoresis in a 2% agarose gel, visualized by ethidium bromide staining,

and the bands were quantitated by densitometry using ImageJ

software.

Western blotFrozen 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 40mg pro-

tein were separated by SDS-PAGE and transferred to a membrane.

After blocking with PBS containing 0.5% skimmed milk, the mem-

brane was incubated at 4�C overnight with primary antibodies: anti-

TNPO3 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-conju-

gated 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).

Anti-TNPO3 and 4’,6-diamidino-2-phenylindole stainingSix-micrometre thick sections of frozen muscle were fixed in ice-cold

acetone for 10min, incubated for 1 h with 1% bovine

serum albumin, and stained at 4�C for overnight with murine anti-

TNPO3 antibody (ab54353, Abcam) at a concentration of 5 mg/ml.

Specimens were then incubated for 1 h with sheep biotinylated anti-

mouse 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 dihy-

drochloride (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).

1510 | Brain 2013: 136; 1508–1517 M. J. Melia et al.

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Results

Clinical assessments

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 distribu-

tion 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 pecu-

liar posture of the feet with elevation of the big toe, foot drop and

weight-bearing on the lateral soles (Supplementary Fig. 2). In add-

ition, 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 Emery-

Dreifuss 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 ex-

ercise 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 atro-

phy 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 asso-

ciated 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 wheel-

chair-bound in the third decade. Two other cases reached Vigno

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 previ-

ously 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 cre-

atine kinase levels (4500U/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 an-

terior (55%), deltoids (46%); (ii) lumbar: paraspinal (90%), ab-

dominal 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 with-

out 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 paraverteb-

ral 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.

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 intermyo-

fibrillar network with abnormal Z bands, rimmed vacuoles and

abnormally increased mitochondria with rare paracrystalline inclu-

sions (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 pa-

tients (Supplementary Fig. 1) confirmed the described abnormal-

ities in myofibres and connective tissue. In addition, we observed

unusually enlarged nuclei with central pallor (Fig. 1). These nuclear


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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 per-

centages were derived from counts of the number of affected

nuclei and the total number of fibres within each biopsy in haema-

toxylin–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 dystro-

phy 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 pseudo-

myelin 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 my-

opathy (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 differ-

ences in DNA from two control subjects and two affected individ-

uals, thereby excluding DNA copy number alterations as the cause

of the disease (data not shown).

Because studies at the DNA level were unrevealing, we per-

formed 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 un-

affected unrelated subjects, using a TaqMan� Custom Array 384-

well microfluidic card (Supplementary Fig. 4 and Supplementary

Table 2). No significant differences could be detected in the ex-

pression of the genes analysed, except for a moderate increase of

Figure 1 Light and electron microscopy findings in muscle biopsies. (A and B) Haematoxylin and eosin staining muscle from affected

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 18–20nm 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: �21000 and �35000. Scale bars = 0.5 mm.


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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; inter-

val: 73.8; 23.9–339.0; n = 5) as compared with levels in un-

affected subjects (RQ median; interval: 3.0; undetectable -10.3;

n = 6). The levels of ADIPOQ transcripts closely correlated with

values for LEP messenger RNA (P40.001; 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 link-

age 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 vari-

ants 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 iso-

forms encoded by the gene. The del-A results in conversion of

TAG to TGC codon, encoding cysteine, and extension of the read-

ing 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 hetero-

zygous 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 pres-

ence 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 segre-

gates 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, comple-

mentary 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 PCR-

amplified. Sequence analysis of this amplified complementary

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 electro-

pherograms (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 individ-

uals 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 termin-

ation 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 signifi-

cant 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 anti-

TNPO3 antibody showed clear nuclear staining and that coloca-

lized with DAPI (Fig. 3I). In muscle of affected individuals, TNPO3

immunostaining was also observed within nuclei, but was un-

evenly distributed and often limited to the periphery of nuclei,

(Fig. 3C and F).

DiscussionLGMD1F is one of the nine autosomal dominant forms of LGMD.

Causative genes had been identified for only five forms of auto-

somal 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 ex-

treme example is LMNA mutations, which have been associated

with a broad spectrum of clinical conditions including Dunnigan

lipodystrophy, autosomal dominant Emery-Dreifuss muscular dys-

trophy, cardiomyopathy, Charcot–Marie–Tooth disease and


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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 + 216bp + 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.


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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 ter-

mination 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 func-

tions, 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 un-

known. TNPO3 was originally identified as TNP-SR2, which en-

codes 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 identi-

fied by genome-wide RNA interference knockdown as a HIV-de-

pendency 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

Figure 3 Anti-TNPO3 and DAPI staining of muscle from affected individuals and controls. Immunofluorescence-stained muscle from two

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 = 40mm.


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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 tran-

scripts 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 mus-

cular 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 con-

tribute 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 pres-

ence of autophagy is due to abnormal protein accumulation,

which confers a dominant toxic function to the autophagic com-

plex that contains the mutated co-chaperone (Sarparanta et al.,

2012). Accordingly, autophagy may be contributing to LGMD1F.

Clinically, LGMD1F was originally described as slowly progres-

sive proximal symmetric weakness with predominantly lower limb

onset, normal to mildly raised creatine kinase activity and myo-

pathic 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 pre-

sent 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 con-

firming the core phenotype of LGMD1F, which is characterized by

pelvic-femoral weakness and less severe and variable shoulder in-

volvement, 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

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 Emery-

Dreifuss 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 know-

ledge 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.

AcknowledgementsThe authors thank the members of the family studied in this work

for their collaboration, and Gisela Nogales-Gadea for scientific

assistance.

FundingThis 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.

Supplementary materialSupplementary material is available at Brain online.

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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



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 desmin-

associated 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

February 12th, 2013


Original Article

Ultrastructural changes in LGMD1F

Giovanna Cenacchi,1 Enrico Peterle,2 Marina Fanin,2 Valentina Papa,1 Roberta Salaroli1 andCorrado Angelini2,3

1Department of Biomedical and Neuromotor Sciences, “Alma Mater” University of Bologna, Bologna, 2Department ofNeurosciences, University of Padova, Padova and 3IRCCS S.Camillo, Venice, Italy

A large Italo-Spanish kindred with autosomal-dominantinheritance has been reported with proximal limb and axialmuscle weakness. Clinical, histological and genetic featureshave been described. A limb girdle muscular dystrophy 1F(LGMD1F) disease locus at chromosome 7q32.1–32.2 hasbeen previously identified. We report a muscle pathologicalstudy of two patients (mother and daughter) from thisfamily. Muscle morphologic findings showed increasedfiber size variability, fiber atrophy, and acid-phosphatase-positive vacuoles. Immunofluorescence against desmin,myotilin, p62 and LC3 showed accumulation of myofibrils,ubiquitin binding protein aggregates and autophagosomes.The ultrastructural study confirmed autophagosomal vacu-oles. Many alterations of myofibrillar component weredetected, such as prominent disarray, rod-like structureswith granular aspect, and occasionally, cytoplasmic bodies.Our ultrastructural data and muscle pathological featuresare peculiar to LGMD1F and support the hypothesis thatthe genetic defect leads to a myopathy phenotype associ-ated with disarrangement of the cytoskeletal network.

Key words: electron microscopy, histopathology, limbgirdle muscular dystrophy 1F, myofibrillar, myopathy.

INTRODUCTION

The limb-girdle muscular dystrophies (LGMDs) are a het-erogeneous group of hereditary neuromuscular disorderswith predominant or selective weakness in the proximallimb and/or distal muscles, having an estimated incidenceof 1:100 000.1–3 The clinical phenotype is characterized by agreat variability, ranging from early onset, with a severeand rapidly progressive clinical course, to milder forms,with a later onset and a slower progression. Autosomal-

dominant (AD) families representing less than 10% of thewhole group of LGMDs,1–3 and to date, eight AD forms ofLGMD, have been described.Among the AD forms, a largeItalo-Spanish kindred with LGMD1F has been describedwith proximal limb and axial muscle weakness.4,5 Clinical,histological and genetic features have been described in5/32 patients. In this family, the disease locus has beenmapped to chromosome 7q32.1–32.2, but no mutationwas detected in filamin C, a possible candidate gene inthis chromosomal region, which encodes for actin bindingprotein highly expressed in muscle.5 The clinical and mor-phological features of two patients with LGMD1F are heredescribed since the disease was not yet sufficiently charac-terized 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 approachhas been performed to investigate the pathogeneticmechanism.

MATERIALS AND METHODS

Muscle biopsy histopathology was investigated in one pairof affected patients (mother, Case 1; daughter, Case 2).Childhood onset was observed in Case 2 (6–7 years) with afaster weakness progression; at 22–23 years the patientshowed difficulty in rising with mild respiratory and swal-lowing impairment. In comparison, in Case 1 clinical symp-toms were relatively mild until the age of 32 years; then theprogression rate was slow. Skeletal muscle biopsies wereperformed in vastus lateralis after obtain patient consent.Muscle specimens were oriented, snap-frozen in liquidnitrogen-chilled isopentane and the cryostat-cut sectionswere stained using a panel of routine histochemicalmethods: HE, modified Gomori trichrome, reducednicotinamide-adenine-dinucleotide-tetrazolium-reductase(NADH-TR), combined cytochrome oxidase (COX) andsuccinic dehydrogenase (SDH), adenosine triphosphatases(ATPases) and acid phosphatase. Immunofluorescenceanalysis for desmin (MAB1698 Chemicon, Temecula, CA,

Correspondence: Giovanna Cenacchi, MD, Department of Biomedi-cal and Neuromotor Sciences, Via Massarenti, 9, 40138 Bologna, Italy.Email: [email protected]

Received 10 July 2012; revised and accepted 8 November 2012.

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Neuropathology 2012; ••, ••–•• doi:10.1111/neup.12003

© 2012 Japanese Society of Neuropathology

December 21st, 2012


US; dilution 1:50) and myotilin (RSO34, Novocastra, New-castle, UK; dilution 1:50) and double-labeling for LC3(2775,Cell SignalingTechnology,Danvers,MA,US;dilution1:100) and p62 (Gp62C, Progen Biotechnik, Heidelberg,Germany; dilution 1:200) were performed using immun-ofluorescence microscopy. Fresh tissues from each biopsywere fixed in 2.5% glutaraldehyde in cacodylate buffer,post-fixed in 1% OsO4 in the same buffer, dehydrated ingraded ethanol, and embedded in araldite. Semithin sec-tions were stained with toluidine blue.Thin sections, stainedwith uranyl acetate and lead citrate, were examined with aPhilips 400T transmission electron microscope.

RESULTS

Morphologic findings by HE showed increased fiber sizevariability; the fibre atrophy was more prominent in Case2, whereas endo- and perimisial connective tissue, and acid-phosphatase-positive areas were more pronounced in Case1 (Fig. 1). Immunofluorescence for desmin and myotilinshowed accumulation of reaction in myofibrillar structuresin some fibers of both patients (Fig. 2). Double-labellingfor p62 and LC3 demonstrated increased protein aggre-gates (p62-positive) within some atrophic fibers (Fig. 3);LC3 labelling, used as a marker of autophagosomes, was

A B

C D

Fig. 1 Muscle biopsy sections stained forHE (A,B), and acid phosphatase (C,D) ofCase 2 (A,C) and Case 1 (B,D). Note fibersize variability, diffuse fiber atrophy inCase 2 (A, B) and accumulations of acidphosphatase-positive material (C,D). Micro-scope magnification ¥200.

Fig. 2 Muscle biopsy sections from Case 2(A,C,D) and Case 1 (B) immunostained fordesmin (A) or myotilin (B–D). Note accumu-lation of cytoskeletal (desmin) or sarcomeric(myotilin) proteins, occupying a relativelylarge cytoplasmic area of isolated myofibers.Microscope magnification ¥200.

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mild and diffuse (Fig. 3). The ultrastructural study con-firmed fiber atrophy (Fig. 4), abnormal mitochondria accu-mulations with only rare paracrystalline-like inclusions(Fig. 4) and autophagosomal vacuoles containing cytoplas-mic debris and myelinoid bodies (Fig. 4). No tubulo-filamentous cytoplasmic or nuclear inclusions weredetected. Many alterations of myofibrillar component wereeasily detected, such as prominent disarray (Fig. 4), rod-like structures with granular aspect (Fig. 4), and occasionalfilamentous cytoplasmic bodies. No ultrastructural differ-ences were appreciated between the two cases.

DISCUSSION

The histopathological data and the electron microscopicfindings from our patients extend previous results describedfor this LGMD1F family.4,5 In the original report, the mor-phologic findings were abnormal fiber size with degenera-tive aspect and prominent rimmed vacuoles.4 COX–SDHstain showed about 5–30% of COX-negative fibres. Theultrastructural description has been performed in threecases.4 That study focused on the presence of bothautophagosomes with cytoplasmic bundles of filaments in

Fig. 3 Double immunofluorescence analy-sis on muscle biopsy sections using anti-bodies against p62 (green) and LC3 (red)(counterstain of nuclei with 4′,6-diamino-2-phenylindole, blue) in Case 2 (A,B) and Case1 (C,D). Note accumulation of p62-positiveprotein aggregates in some atrophic musclefibers in both patients.

A B

C D

Fig. 4 Ultrastructural analysis showingatrophic fiber characterized by many poly-morphic mitochondria with paracrystalline-like inclusions (arrow) (A) and myelinoidbody adjacent to the subsarcolemmalnucleus (B) in Case 2. Myofibrillar altera-tions lead to architectural disarray of musclefibers in Case 1 (C), and to accumulation ofelectrondense material of possible Z-line-derivation in the subsarcolemmal areas inCase 2 (D). At higher magnification, theelectron-dense material appears as agranulo-filamentous pattern (d-inset). Scalebar = (A) 2000 nm; (B) 1000 nm; (C)2000 nm; (D) 5000 nm (inset: 2000 nm).

Ultrastructure of LGMD1F 3


December 21st, 2012


one case,and a large amount of degenerating mitochondria,which were considered a secondary unspecific feature.4

Changes related to mitochondria and autophagosomalvacuoles are commonly seen in a wide variety of myopathiessuch as inclusion body myositis (IBM)6,7 and oculopharyn-geal nuscular dystrophy (OPMD).8–10 Whereas abnormali-ties of p62 and ubiquitin-binding proteins might be signalsof protein degradation, in our cases protein aggregates wereassociated with p62 and diffuse LC3. Abnormalities ofNBR1 (neighbor of BRCA1 gene 1), a novel autophagy-associated protein, could be a useful tool to further studychronic progressive myopathies.7 We did not identify in ourpatients’ biopsies, neither nuclear nor cytoplasmic tubulo-filament inclusions, which are considered specific for IBM ifassociated with the presence of rimmed vacuoles.6,7 We didnot see intranuclear tubulofilaments arranged in tangles orpalisades which are described in the OPMD.9,10 The presentultrastructural observations highlight in both cases severemodifications of myofibrillar filaments which appeardisorganized with granular rod-like structures, cytoplasmicbodies and myofibrillar disarray with focal perpendiculararrangement. The presence of myofibrillar disarray andgranulo-filamentous material is likely to derive from theZ-line, and this is supported by immunofluorescence fordesmin and myotilin, which are usually observed in myofi-brillar myopathies. Among myopathies characterized bymyofibrillar derangement, the myofibrillar myopathieshave been well described.11–14 They are a group of muscledisorders associated with similar morphologic featuresconsisting of myofibrillar disorganization originating fromthe Z-disk followed by accumulation of myofibrillar degra-dation products. They may be defined by the presenceof rimmed vacuoles, associated with ectopic expressionof multiple proteins that include desmin, neural celladhesion molecule, plectin, gelsolin, ubiquitin, Xin, TARDNA-binding protein 43 and cochaperones, includingaB-crystallin, heat shock protein-27.11–14 Myotilinopathy(LGMD1A) and filaminopathy have been reported as asubset of myofibrillar myopathy. Both myotilinopathy andfilaminopathy (the so-called Z-disk diseases) exhibit themorphological findings typical of myofibrillar myopathywith filament accumulation including Z-disk alterations.12,13

Particularly in filaminopathy,strongly positive for filamin C,ultrastructural examination revealed major myofibrillarabnormalities, including accumulation of desmin-positivegranulo-filamentous material. In addition, also largeautophagic vacuoles and mitochondrial aggregates in theabnormal fiber regions were observed.15–17 The diseasemechanism in filaminopathy is still unclear, but it mayinvolve structural alterations of the Z-disk caused by dys-functional proteins or their abnormal accumulation due todefective degradation.12,13 In particular, among cytoskeletalproteins, desmin, with its binding partners, forms a three-

dimensional scaffold around Z-disks, thereby interlinkingwith myofibrils and nuclei, mitochondria and sarcolemma.Several studies demonstrated that the filamentous desminnetwork plays an essential role in the subcellular position-ing and function of mitochondria.11–14 Indeed,mitochondrialaccumulation has been clearly showed in LGMD1F, and itwas confirmed also in the present two cases, where rareparacrystalline inclusions were found similar to those pre-viously described.4

The contemporary presence of autophagosomes andseveral myelin figures is the morphological substrateof a protein quality-control disturbance related to theubiquitin–proteasome system (UPS) and the autophagic-lysosomal pathway.11 A recent study showed that desminmutants impair the proteolytic function of the UPS and theautophagic–lysosomal pathway (types 1 and 2 of pro-grammed cell death).11

Electron microscopy is useful in the diagnostic workupof chronic myopathies identifying pathological proteinaggregation, cytoplasmic/spheroid bodies, and signs ofmyofibrillar degeneration, such as sarcoplasmic granulo-filamentous material, autophagic vacuoles and myelin-likewhorls.11 To address the alteration of the Z-line,both degen-eration, streaming, irregularities and Z-line loss should bedetected. Furthermore, additional features can be found,such as depletion or accumulation of mitochondria.

Our morphological and ultrastructural data seem tosuggest in our LGMD1F cases a myopathy phenotypesimilar to those described for Z-disk diseases.Although thegenetic defect is still under investigation, it is possible tohypothesize that the mutant protein in LGMD1F mightlead to disarrangement of desmin-associated cytoskeletalnetworks.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

REFERENCES

1. Broglio L, Tentorio M, Cotelli MS et al. Limb-girdlemuscular dystrophy-associated protein diseases. Neu-rologist 2010; 16: 340–352.

2. Guglieri M, Straub V, Bushby K, Lochmuller H. Limb-girdle muscular dystrophies. Curr Opin Neurol 2008;21: 576–584.

3. Nigro V, Aurino S, Piluso G. Limb girdle musculardystrophies: update on genetic diagnosis and therapeu-tic approaches. Curr Opin Neurol 2011; 24: 429–436.

4. Gamez J, Navarro C, Andreu AL et al. Autosomaldominant limb-girdle muscular dystrophy. A largekindred with evidence for anticipation. Neurology2001; 56: 450–454.

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5. Palenzuela L, Andreu AL, Gàmez J et al. A novelautosomal dominant limb-girdle muscular dystrophy(LGMD1F) maps to 7q32.1-32.2. Neurology 2003; 61:404–406.

6. Askanas V, Engel WK. Sporadic inclusion-bodymyositis: conformational multifactorial ageing-relateddegenerative muscle disease associated with proteaso-mal and lysosomal inhibition, endoplasmic reticulumstress, and accumulation of amyloid-b42 oligomers andphosphorylated tau. Presse Med 2011; 40: 219–235.

7. D’Agostino C, Nogalska A, Cacciottolo M, Engel WK,Askanas V. Abnormalities of NBR1, a novelautophagy-associated protein, in muscle fibers of spo-radic inclusion-body myositis. Acta Neuropathol 2011;122: 627–636.

8. Gambelli S, Malandrini A, Ginanneschi F et al. Mito-chondrial abnormalities in genetically assessed oculo-pharyngeal muscular dystrophy. Eur Neurol 2004; 51:144–147.

9. Van Der Sluijs BM, Hoefsloot LH, Padberg GW, VanDer Maarel SM, Van Engelen BG. Oculopharyngealmuscular dystrophy with limb girdle weakness asmajor complaint. J Neurol 2003; 250: 1307–1129.

10. Schröder JM, Klossok T, Weis J. Oculopharyn-geal muscle dystrophy: fine structure and mRNA

expression levels of PABPN1. Clin Neuropathol 2011;30: 94–103.

11. Schröder R, Schoser B. Myofibrillar myopathies: aclinical and myopathological guide. Brain Pathol 2009;19: 483–492.

12. Selcen D. Myofibrillar myopathies. Curr Opin Neurol2010; 23: 477–481.

13. Selcen D. Myofibrillar myopathies. NeuromusculDisord 2011; 21: 161–171.

14. Montse O, Odgerel Z, Martınez A et al. Clinical andmyopathological evaluation of early- and late-onsetsubtypes of myofibrillar myopathy. NeuromusculDisord 2011; 21: 533–542.

15. Vorgerd M, van der Ven PFM, Bruchertseifer V et al. AMutation in the dimerization domain of Filamin Ccauses a novel type of autosomal dominant myofibril-lar myopathy. Am J Hum Genet 2005; 77: 297–304.

16. Kley RA, Hellenbroich Y, van der Ven PFM et al.Clinical and morphological phenotype of the filaminmyopathy: a study of 31 German patients. Brain 2007;130: 3250–3264.

17. Shatunov A, Olive M, Odgerel Z et al. In-frame dele-tion in the seventh immunoglobulin-like repeat offilamin C in a family with myofibrillar Myopathy. Eur JHum Genet 2009; 17: 656–663.

Ultrastructure of LGMD1F 5


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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 X-

linked recessive mutations in dystrophin, with only rare reports of asymptomatic adult males. The static cognitive impairment seen in dystro-phinopathies is thought to be due to altered expression of dystrophin iso-forms, 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

1Seconda Universita degli Studi di Napoli, Laboratorio di Genetica Medica,

Dipartimento di Patologia Generale, Napoli, Italy; 2TIGEM, Telethon

Institute of Genetic and Medicine, Napoli, Italy; 3Seconda Universita degli

Studi di Napoli, Dipartimento di Patologia Generale, Napoli, Italy; 4Secon-

da Universita degli Studi di Napoli, Cardiomiologia e Genetica Medica,

Napoli, Italy; 5Universita degli Studi di Padova, Department of Neurosci-

ences, Padova, Italy

Next generation sequencing (NGS) is having a tremendous impact on

our knowledge of different aspects of biology. It can be also very pow-

erful to study patients with heterogeneous genetic conditions, like muscu-

lar 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 sep-

arated by up to eleven meioses and identified a single shared novel het-

erozygous frame-shift variant. This causes a nonstop change in the

Transportin 3 (TNPO3) gene that encodes a member of the importin-bsuper-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 SureSe-

lect 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 11IGBMC, Translational Medecine, Illkirch, France; 2 IGBMC, Illkirch,

France; 3 Insitut de Myologie, Unite de Morphologie Neuromusculaire, Paris, France; 4 Faculte de Medecine 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 disease-causing mutations in RYR1 and NEB genes, large genes not screened on rou-tine if RNA is not available. Phenotypes of patients with RYR1 muta-

tions 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

806 Abstracts / Neuromuscular Disorders 22 (2012) 804–908

August 30th, 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 leftvastus 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 atrophyor 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 sub-group 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.

June 12th, 2012


P115

IDENTIFICAZIONE DI NUOVI GENI COINVOLTI NELLE DISTROFIE MUSCOLARI DEI CINGOLI MEDIANTE ARRAYSE SEQUENZIAMENTO DI NUOVA GENERAZIONE (NGS)

A. Torella 1, F. Del Vecchio Blanco 3, M. Dionisi 2, A. Garofalo 3, M. Iacomino 3, M. Mutarelli 2, M. Savarese 1, G. Piluso1, V. Nigro 11Dip. di Patologia Generale-Lab. di Genetica Medica, Seconda Università degli Studi di Napoli, Telethon Institute ofGenetics and Medicine (TIGEM)2Telethon Institute of Genetics and Medicine (TIGEM)3Dip. di Patologia Generale-Lab. di Genetica Medica, Seconda Università degli Studi di NapoliCampioni 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 sarcoglicanie un altro 10% negli altri geni noti LGMD:LGMD2A-N. Una significativa percentuale di pazienti con LGMD non avevaalcuna 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 diconcentrare la ricerca delle mutazioni su un singolo gene alla volta. Inoltre, gli attuali esami genetici sono lunghi, costosi esenza effetti. I nuovi potenti approcci per lʼanalisi del DNA, come la next-generation sequencing (NGS) sono in procinto dirivoluzionare 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 molecolarinon 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 copertu-ra esonica completa dei geni coinvolti nelle malattie neuromuscolari che permette di individuare delezioni o duplicazionideleterie. Gli esomi di 16 soggetti appartenenti a 7 diverse famiglie sono stati sequenziati mediante NGS utilizzando lapiattaforma 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 (fra-me-shift) non trovata negli altri membri della famiglia o controlli. In una seconda famiglia LGMD con ereditarietà AR abbia-mo recentemente identificato una mutazione missenso in omozigosi nel gene ACADVL che è condivisa da tutti i membriaffetti della famiglia e da altri pazienti provenienti dalla stessa area geografica.

2012


168

Acta Myologica • 2011; XXX: p. 168

ADDENDUM

LGMD 1(F) - A pathogenetic hypothesis based on histopathology and ultrastructureG. Cenacchi, E. Peterle, L. Tarantino, V. Papa, M. Fanin,C. AngeliniClinic Department of Radiologic and histopathologic Sciences,University of Bologna, Department of Neurosciences andVIMMM, University of Padua

A large Spanish kindred with apparently autosomal domi-nant 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 ex-pressed in muscle. We report a clinico-pathological study of two

Proceedings of the XI Congress of the Italian Association

of MyologyCagliari, May 2011

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 antici-pation. 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 vac-uoles. The ultrastructural study confirmed fiber atrophy, abnor-mal 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 eas-ily detected such as prominent disarray, rod-like structures with granular aspect, and occasionally cytoplasmic bodies. Our mor-phological data support the hypothesis that other actin-encoding proteins such as FSCN3, and KIAA0265 from the same criti-cal region may represent attractive candidate genes in the LG-MD 1(F) pathogenetic mechanism.

Abstract omitted in Acta Myologica, Vol. XXX, June 2011

October 30th, 2011


Autosomal dominant limb-girdlemuscular 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 beenidentified, including five types of autosomal dominant LGMD (AD-LGMD). Objective: To describe clinical, histologic, andgenetic features of a large Spanish kindred with LGMD and apparent autosomal dominant inheritance spanning fivegenerations. 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 individualshad weakness of the pelvic and shoulder girdles. Severity appeared to worsen in successive generations. Muscle biopsyfindings were nonspecific and compatible with MD. Linkage analysis to chromosomes 5q31, 1q11-q21, 3p25, 6q23, and 7qdemonstrated that this disease is not allelic to LGMD forms 1A, 1B, 1C, 1D, and 1E. Conclusions: This family has a geneticallydistinct 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) com-prise a genetically diverse group of muscle disorderswith predominantly proximal limb and axial muscleweakness. Because of molecular genetic discoveriesand improved clinical criteria, the classification andnomenclature of LGMD have evolved over the lastdecade.1,2 Many LGMD disorders are autosomal re-cessive traits, but at least five well-characterizedforms have been reported in recent years.3-12

We studied a large Spanish kindred with 32 af-fected individuals and apparently autosomal domi-nant inheritance spanning five generations. Here, wedescribe the clinical phenotype and morphologic find-ings in five patients who underwent muscle biopsy,and preliminary genetic investigations of this newtype of autosomal dominant LGMD (AD-LGMD).

Patients and methods. A total of 61 individuals fromfive generations of a family from eastern Spain were exam-ined. Serum creatine kinase (CK), aspartate aminotrans-ferase (ASAT), and alanine aminotransferase (ALAT)determinations were performed on all subjects. Twelve un-derwent neurophysiologic examinations, and five hadmuscle biopsy. Other investigations included electrocar-diography (ECG) in 12 patients, echocardiography in sixpatients, and MRI of the brain in two patients.

Subjects were considered affected when clinical exami-

nation revealed a characteristic pattern of muscular weak-ness, primarily affecting the pelvic and shoulder girdles.Muscle strength was assessed using the British MedicalResearch Council (MRC) Scale; 26 muscle groups were ex-amined bilaterally. Functional ability was measured ac-cording to the scales designed by Vignos and Brooke.13-14

Age at onset was determined using a standardized clinicalquestionnaire form asking clinically affected individuals toidentify their first symptoms from a list that included awaddling gait and difficulty in climbing stairs, raisinghands above the head, lifting, running, and rising from achair or squatting position. A total of 32 family memberswere clinically affected. In figure 1, individuals are identifiedby generation number (Roman numerals) followed by birthorder position, reading from left to right (Arabic numerals).

Skeletal muscle biopsy specimens from the deltoid orvastus lateralis were oriented, snap-frozen in liquidnitrogen-chilled isopentane and the cryostat-cut sectionswere stained using standard histochemical methods. Im-munohistochemistry was performed using the followingantibodies: desmin and vimentin (Biogenex, CA), ubiquitin(DAKO, DK), Tau protein and Beta-amyloid (Sigma, MO),and dystrophin, sarcoglycans, and the amino terminal ofutrophin (DRP2, Novocastra Laboratories, Newcastle uponTyne, UK). A small portion of each sample was fixed inglutaraldehyde and processed for ultrastructuralexamination.

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 ResearchCenter for Muscular Dystrophy and Related Diseases (Drs. Karadimas, DiMauro, and Hirano), Department of Neurology, Columbia University College ofPhysicians 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.

August 8th, 2000


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 thepedigree after informed consent. After DNA extractionfrom the blood buffy coats, a set of fluorescent-labeled mi-crosatellite simple sequence repeat (SSR) markers span-ning the five known loci of LGMD1 were genotyped usingan ABI Prism 310 Genetic Analyzer (Perkin Elmer, FosterCity, CA).

In addition, SSR markers encompassing the loci of auto-somal recessive HIBM (AR-HIBM),15 autosomal dominantHIBM (AD-HIBM),16 Bethlem myopathy,17-18 and fa-cioscapulohumeral dystrophy (FSHD)19 were genotyped.

These SSR markers were as follows:1. LGMD 1A (5q31): D5S410, D5S436, D5S2115, and

D5S4712. LGMD 1B (1q11-q21): D1S218, D1S196, D1S2878,

D1S484, D1S2635, D1S498, D1S252, and D1S27263. LGMD 1C (3p25): D3S1277, D3S1266, D3S2338,

D3S1263, D3S1304, and D3S12974. LGMD 1D (6q23): D6S308, D6S292, D6S262, and

D6S2875. LGMD 1E (7q): D7S427, D7S2465, D7S798, D7S636,

and D7S6616. AR-HIBM (9p1-q1): D9S161, D9S1817, D9S273, and

D9S1757. AD-HIBM (17p13.1): D17S938, D17S1852, D17S799,

and D17S9218. Bethlem myopathy (2q37 and 21q22.3); D2S206,

D2S338, D2S125, D21S1252, D21S1255, and D21S2669. FSHD (4q35): D4S2920, D4S1535, D4S2924, and

D4S426Two-point (marker-to-disease) analysis was performed

with the MLINK option of FASTLINK 4.0.20

The disease was considered autosomal dominant with90% penetrance. The disease gene frequency was esti-mated at 1/100,000. Marker allele frequencies were esti-mated by allele counting of all genotyped subjects. TheLINKMAP option was used for three-point analyses.Marker order and intermarker distances were based onthe Genethon linkage map.

Results. Characteristic muscle weakness predominantlyinvolving the pelvic and shoulder girdle proximal muscleswas shown in 32 individuals (15 men, 17 women) betweenthe ages of 7 and 66 years (mean 34.5, SD � 14.4 years).

Age at onset ranged from less than 1 to 58 years (mean16.3, SD � 15.5 years). Two groups were delineated basedon age at onset: a juvenile form, with onset before age 15(65.6% of patients) and an adult-onset form, startingaround the third or fourth decade (28.0%).

Symptoms of pelvic girdle muscular weakness werenoted at onset in 81.1% of cases. Commonly affected mus-cles were the iliopsoas (96.0%), gluteal (75.0%), hip adduc-tors (71.8%), deltoid (90.6%), biceps brachii (68.7%),paraspinal (65.6%), and neck flexors (62.5%). Pelvic girdleimpairment was more severe and occurred earlier than inthe shoulder girdle. Proximal muscle weakness rangedfrom MRC grade 0 to 4�/5, with symmetric distribution.

Distal weakness appeared late in the disease’s course oraccompanied initial presentation in severely affectedjuvenile-onset patients, frequently affecting the extensordigitorum, tibialis anterior, and toe extensor muscles.

Six patients had scapular winging. Two juvenile-onsetpatients showed mild facial weakness 10 years after onset.Early-onset patients had generalized muscular wasting, pre-dominantly involving the quadriceps, gluteus, deltoid, biceps,infraspinatus, and supraspinatus muscles (figure 2).

Early joint contractures were not present. Three indi-viduals developed Achilles tendon contractures late in thedisease’s course. Four subjects showed scoliosis or hyper-lordosis. All belonged to the juvenile-onset group.

Respiratory muscles were clinically affected in four pa-tients with juvenile-onset form. Mean forced vital capacityin these patients was 38.6% of predicted values.

No patient had ptosis, ophthalmoparesis, dysphagia,speech disturbances, calf hypertrophy, myalgia, or intellec-tual deterioration.

Weakness progress during the 8-year follow-up showedtwo patterns: relatively slow in adult-onset subjects, andfaster in juvenile-onset subjects. The progression rateseemed linear. Two patients with onset before age 14 be-came wheelchair-bound before age 28. A boy with onset atage 1 year needed assistance walking by age 12, and his

Figure 1. Drawing of the pedigree. Clinically affected members are shown in black. Roman numerals indicate generationnumber, and Arabic numerals birth order position within the generation.

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mother’s help with most everyday tasks (Vignos’ scalegrade 6).

Mean CK was 589, ranging between 47 and 2,920 (nor-mal �150 U/L). Normal CK was recorded in 40.6% of pa-tients. Mean ASAT and ALAT were 35.7 and 44.5 U/L.

Electromyography (EMG) showed myopathic changeswith short duration, polyphasia, and low-amplitude poten-

tials, which were more pronounced in the proximal mus-cles. Sensory and motor nerve conduction velocities werenormal.

Brain MRI, ECG, and echocardiograms, when per-formed, were normal.

The inheritance pattern is consistent with autosomaldominant transmission; 44 of 76 (58%) children of affectedparents manifested the disease. We identified 26 affectedparent/affected child pairs. The pair-wise data comprises18 pairs with a generation III parent and eight pairs witha generation IV parent. In the generation III parent pairs,we observed a mean decrease of 28.5 years in an offspring’sonset age. In the generation IV parent pairs, the meandecrease in an offspring’s onset age was 13.2 years. Thus,apparent anticipation was significant (p � 0.000 in gener-ation III parents and p � 0.002 in generation IV parents).Overall comparison of age at onset curves in life tableanalysis between patients of generations III, IV, and Vshowed a decrease in age at onset (Wilcoxon test statistic� 16.84, p � 0.0002). We also examined the effects ofparental gender origin on anticipation. The mean differ-ence in age at onset between parent and child in 17 moth-er/child pairs was 1.6 years younger than in 9 father/childpairs (two-tailed Mann–Whitney U test � 59.5, p � 0.367).In our data, no evidence suggests a significant effect ofparental gender on age at onset.

Muscle biopsy. Light microscopy. Open muscle bi-opsy was performed on patients III-8, IV-6, IV-11, IV-21,and V-11. When biopsied, their ages varied between 9 and59 years (mean 31.4 years). Morphologic findings weresimilar in all cases, composed of abnormal fiber size andshape variation, increased endo- and perimysial connectivetissue, scattered degenerative fibers with myophagia, ab-normal Z-bands, and, in three of five cases, prominentrimmed vacuoles (figure 3A). Central nuclei were occasion-ally present. Fiber type differentiation and distributionwere normal. One patient (V-11) showed a significantnumber of ragged-red fibers (RRF) (�15%); cytochrome coxidase (COX)–succinate dehydrogenase technique dis-closed between 5 and 30% COX-negative fibers in three offive cases. All RRF were COX-negative, but COX-negativefibers without signs of mitochondrial proliferation werealso present. Immunohistochemical stains for dystrophinand sarcoglycans were normal. No abnormal deposits oftau, ubiquitin, or �-amyloid proteins were found. Desminwas overexpressed in some fibers, but was not abundantenough for consideration as a significantly abnormaldesmin accumulation. Vimentin and dystrophin-relatedprotein overexpression in scattered small fibers without

Figure 2. Patient showing lordosis, scapular winging,proximal wasting affecting the pelvic and shoulder mus-cles, and a sparing of the facial muscles.

Figure 3. (A) Muscle cryostat section inSubject IV-6. Notice increased fiber sizevariability, one small fiber with twoprominent rimmed vacuoles (bottomright corner) and a fiber with subsar-colemmal basophilia and marked inter-myofibrillary network, indicative ofmitochondrial proliferation (center) (he-matoxylin– eosin �60 before reduction).(B) Electron micrograph in SubjectV-11. Skeletal muscle fiber cut trans-

versally. Notice increased number of paranuclear mitochondria with abnormal cristae and paracrystalline inclusions(�8,000 before reduction).

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dystrophin deficiency was considered evidence ofregeneration.

Electron microscopy. Three cases showed autophagicvacuoles with prominent pseudomyelin figures and densebodies. One case (IV-6) showed intracytoplasmic bundles of16- to 18-nm filaments. Despite thorough searching, noadditional filamentous inclusions were observed in the nu-clei or in the other four patients’ cytoplasm or nuclei. Onepatient (V-11) showed prominent subsarcolemmal andparanuclear abnormal mitochondria accumulations, manywith paracrystalline inclusions (see figure 3B).

Linkage analysis. Using genotype data of SSR mark-ers, three-point analyses excluded linkage of the LGMD inour pedigree to known chromosomal loci for the following:LGMD forms 1A, 1B, 1C, 1D, and 1E, autosomal dominantand recessive HIBM, Bethlem myopathy, autosomal domi-nant Emery–Dreifuss dystrophy (AD-EDMD), and FSHDbased on three-point lod scores ��2.0.

Discussion. Thirty-two patients were identifiedfrom a large Spanish family with autosomal domi-nant muscular dystrophy. The disorder fulfillsLGMD diagnostic criteria,21 with the following clini-cal features: slowly progressive proximal symmetricweakness with predominantly lower limb onset, nor-mal to mildly raised CK activity, myopathic EMGand muscle biopsy changes, and normal skeletalmuscle dystrophy staining.

Variability was observed in age at onset, muscularsymptomatology, and progression rates, with two pa-tient groups differentiated by severity and progres-sion: an adult-onset form, around the fourth decade,and a juvenile form, beginning before age 15 years.Patients with severe functional disability belonged tothe second group.

Dysarthria, cardiac involvement, calf hypertro-phy, and contractures are features of other AD-LGMD forms, not seen in our patients. Anotherfeature of our family was an early onset age (mean16.3 years). In nearly two thirds of patients, onsetoccurred in childhood or adolescence. Patients withonset before 12 years of age presented with general-ized muscular weakness.

We noted an anticipation phenomenon in 26 parent–child pairs, with the parents two and three genera-tions removed from a common ancestor. Earlieronset in children than in parents suggested geneticanticipation. Disease severity seems unrelated to thetransmitting parent’s gender. Anticipation phenome-non was described in LGMD-1.6

The absence of facial, bulbar, or cardiac impair-ment excluded other myopathies. The lack of earlyjoint contractures suggests our AD-LGMD pedigreediffers from Bethlem myopathy or AD-EDMD. Weexcluded linkage to chromosomal loci for Bethlemmyopathy and AD-EDMD.17,18,22-24

Some patients with FSHD lack facial involvement,thus resembling LGMD. However, in our family theabsence of shoulder girdle muscle weakness asym-metry, predominant muscle weakness of scapularfixators, hearing loss, and facial weakness affectingeye 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 link-age of the diseases to the 4q35 FSHD locus.19

Muscle biopsies showed rimmed vacuoles in threepatients. Initially, a diagnosis of HIBM was consid-ered because of this and the presence of filamentousinclusions.25

Most HIBM cases reported have shown autosomalrecessive inheritance26-27 but AD-HIBM has beenrarely described.28-29

However, rimmed vacuoles without the character-istic filaments are frequent nonspecific findings indifferent muscle disorders, including forms ofLGMD17,30 and primary dystrophinopathies.31 We ex-cluded linkage to the identified loci for the identifiedloci of autosomal recessive and dominant HIBM.15-16

We interpreted the mitochondrial proliferation inone patient’s muscle as a secondary phenomenon.

Five AD-LGMD (LGMD1) forms have so far beendelineated.2,5,8-12 Types 1B and 1E are associatedwith cardiologic abnormalities, including atrioven-tricular conduction disturbances, arrhythmias andsudden death. Type 1A is characterized by a dysar-thric 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 thoseloci found no association, suggesting a geneticallydistinct disorder. We are performing a genome-widescan to identify the disease locus.

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