Aiutaci a camminare, aiutaci a vivere. Insieme possiamo farcela!

Transcription

A S S O C I A Z I O N E
Aiutaci a camminare, aiutaci a vivere.
Insieme possiamo farcela!
Paraje de Reolid 2, 30420 Valentín, Murcia, España
C.F.G73886483
Email: [email protected]
Web: www.conquistandoescalones.org
Facebook: www.facebook.com/conquistandoescalonesita
Twitter: @AceDistrofiaIT
Youtube: Conquistando Escalones Italia
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.....................22
Marina Fanin, PhD1, Enrico Peterle, MD1, Chiara Fritegotto, PhD1, Anna C. Nascimbeni,
PhD1, Elisabetta Tasca, PhD2, Annalaura Torella, PhD3,4, Vincenzo Nigro, MD, PhD3,4,
Corrado Angelini, MD1,2
•
Genetic basis of limb-girdle muscular dystrophies: the 2014 update..................24
•
P.5.10 - Clinical and ultrastructural changes in transportinopathy......................36
•
P.5.12 - A mutation in TNPO3 causes LGMD1F and characteristic nuclear
pathology............................................................................................................. 37
Vincenzo Nigro e Marco Savarese
C. Angelini 1, E. Peterle 1, M. Fanin 1, G. Cenacchi 2, V. Nigro 3
A. Kubota 1, M.J. Melia 2, S. Ortolano 3, J.J. Vilchez 4, J. Gamez 5, K. Tanji 6, E. Bonilla 6,
L. Palenzuela 2, I. Fernandez-Cadenas 2, A. Pristoupilova 7, E. Garcia-Arumi 2, A.L.
Andreu 2, C. Navarro 3, R. Marti 2, M. Hirano 1
•
Distrofia dei cingoli, Telethon scopre il gene responsabile della rara patologia..38
•
Next-Generation Sequencing Identifies Transportin 3 as the Causative Gene for
LGMD1F.............................................................................................................. 39
Annalaura Torella1,2, Marina Fanin3, Margherita Mutarelli1, Enrico Peterle3, Francesca Del
Vecchio Blanco2, Rossella Rispoli1,4, Marco Savarese1,2, Arcomaria Garofalo2, Giulio
Piluso2, Lucia Morandi5, Giulia Ricci6, Gabriele Siciliano6, Corrado Angelini3,7, Vincenzo
Nigro1,2
•
Clinical phenotype, muscle MRI and muscle pathology of LGMD1F..................46
•
Limb-girdle muscular dystrophy 1F is caused by a microdeletion in the
transportin 3 gene................................................................................................55
Enrico Peterle1 , Marina Fanin1 , Claudio Semplicini1 , Juan Jesus Vilchez Padilla2 ,
Vincenzo Nigro3,4 , Corrado Angelini1,5
Maria J. Melià1,2, Akatsuki Kubota3, Saida Ortolano4, Juan J. Vilchez5, Josep Gámez6,
Kurenai Tanji7, Eduardo Bonilla3,7,†, Lluis Palenzuela1,2, Israel Fernandez-Cadenas1, Anna
Pristoupilová8,9, Elena Garcia-Arumí1,2, Antoni L. Andreu1,2, Carmen Navarro2,4, Michio
Hirano3,and Ramon Marti1,2
•
P07 Limb-Girdle Muscular Dystrophy and Inherited Myopathy Limb Girdle
Muscular dystrophy 1F: Clinical, Molecular and Ultrastructural study (P07.032)
............................................................................................................................. 65
Corrado Angelini1, Enrico Peterle2, Marina Fanin3, Giovanna Cenacchi4 and Vincenzo
Nigro5
•
Ultrastructural changes in LGMD1F.................................................................... 66
•
D.O.3 Next generation sequencing application are ready for genetic diagnosis of
muscular dystrophies...........................................................................................71
Giovanna Cenacchi1, Enrico Peterle2, Marina Fanin2, Valentina Papa1, Roberta Salaroli1
and Corrado Angelini2,3
M. Savarese 1, A. Torella 1, M. Mutarelli 2, M. Dionisi 2, T. Giugliano 3, G. Di Fruscio 3, M.
Iacomino 3, A. Garofalo 3, S. Aurino 3, F. Del Vecchio Blanco 3, G. Piluso 3, L. Politano 4,
M. Fanin 5, C. Angelini 5, V. Nigro 3
2
•
New quantitative MRI indexes useful to investigate muscle disease..................72
•
Identificazione di nuovi geni coinvolti nelle distrofie muscolari dei cingoli
mediante arrays e sequenziamento di nuova generazione (NGS).....................73
C. Angelini, M. Fanin, E. Peterle
A. Torella 1, F. Del Vecchio Blanco 3, M. Dionisi 2, A. Garofalo 3, M. Iacomino 3, M. Mutarelli
2
, M. Savarese 1, G. Piluso 1, V. Nigro 1
•
LGMD 1(F) - A pathogenetic hypotesis based on histopathology and
ultrastructure........................................................................................................ 74
G. Cenacchi, E. Peterle, L. Tarantino, V. Papa, M. Fanin, C. Angelini
•
A novel autosomal dominant limb-girdle muscular dystrophy (LGMD 1F) maps to
7q32.1-32.2..........................................................................................................75
L.Palenzuela1, PhD; A.L.Andreu1, MD, PhD; J.Gámez2, MD, PhD; M.R.Vilà3, PhD;
T.Kumimatsu3, PhD; A.Meseguer1, PhD; C.Cervera2, MD, PhD; I.Fernández Cadenas1,
Msc; P.F.M. Van der Ven4, PhD; T.G.Nygaard5, MD; E.Bonilla3, MD; and M. Hirano3, MD
•
Autosomal dominant limb-girdle muscular dystrophy..........................................78
J. Gamez1, MD; C. Navarro 3, MD; A.L. Andreu2, MD; J.M. Fernandez4, MD; L.
Palenzuela2, MS; S. Tejeira3, MS; R. Fernandez–Hojas3, MS; S. Schwartz2, MD, PhD; C.
Karadimas5, PhD; S. DiMauro5, MD; M. Hirano5, MD; and C. Cervera1, MD
3
Che cos’è A.C.E.?
4
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.
8
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!
9
Riassunto e traduzione delle pubblicazioni
10
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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,
1.
2.
3.
4.
Dipartimento di Neuroscienze, Università di Padova, Padova, Italia
IRCCS Fondazione San Camillo, Venezia, Italia
Dipartimento di Biochimica, Biofisica e Patologia Generale, II Università di Napoli, Napoli, Italia
Istituto Telethon di Genetica e Medicina (TIGEM), Napoli, Italia
La Distrofia Muscolare dei Cingoli (LGMD) tipo 1F (MIM #608423) è una rara patologia
autosomica dominante il cui locus è stato mappato ed è stato identificato il gene a
seguito di una ricerca in una stessa numerosa famiglia. Durante l’esame di questa
famiglia, si è ampliata la genealogia iniziale e le caratteristiche cliniche della malattia. Si
caratterizza per un grado variabile di debolezza muscolare e limitazione funzionale, con
un 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 alcuni
pazienti, la malattia si trasmette attraverso genitori apparentemente non colpiti
(penetranza incompleta). Per calcolare il tasso di penetranza esatto, si è esaminato
tanto il fenotipo clinico quanto il genotipo di 115 membri della famiglia. I risultati ottenuti
possono essere utili per una consulenza genetica, specialmente per i pazienti più
giovani in cui la mutazione è presente.
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Vincenzo Nigro e Marco Savarese
Dipartimento di Biochimica, Biofisica e Patologia Generale, Seconda Università degli Studi di Napoli e
Istituto Telethon di Genetica e Medicina (TIGEM), Napoli, Italia
Le distrofie muscolari dei cingoli (LGMD) sono un gruppo di alterazioni muscolari
altamente eterogenee, che inizialmente colpiscono i muscoli volontari dell’area dei
cingoli pelvico e scapolare. La definizione è molto descrittiva e meno ambigua per
esclusione: non X linked, non FSH, non miotonica, non distale, non sindromica e non
congenita. Attualmente, la classificazione genetica sta diventando troppo complessa
dato che l’acronimo LGMD è stato utilizzato anche per altre patologie muscolari con
fenotipi sovrapposti.
Attualmente, la lista di geni da rivedere è troppo vasta per un approccio gene per gene
ed è più adatta per essere mirata a pannelli di Next Generation Sequencing (NGS) che
dovrebbero includere qualsiasi gene che fino adesso sia stato associato al quadro
clinico di LGMD.
Il presente articolo ha lo scopo di riassumere la base genetica delle LGMD ordinando e
proponendo una nomenclatura per le forme orfane. Questo è utile visto il ritmo delle
nuove scoperte.
Fino ad oggi si sono identificati trentun loci, 8 autosomici dominanti e 23 autosomici
recessivi. Le forme dominanti (LGMD1) sono: LGMD1A (myotilin), LGMD1B (lamin A/C),
LGMD1C (caveolin3), LGMD1D (DNAJB6), LGMD1E (desmin), LGMD1
F(
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, LGMD1G (HNRPDL), LGMD1H (chr. 3). Le forme autosomiche recessive (LGMD2)
sono LGMD2A (calpain3), LGMD2B (dysferlin), LGMD2C (γ sarcoglycan), LGMD2D (α
11
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 aut
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LGMD1
Fè stata mappata originariamente in un intervallo di 3.68 Mb nel cromosoma
7q32.1-7q32.2 in una numerosa famiglia italo-spagnola. Abbiamo presentato
l’identificazione di TNPO3 mediante un sequenziamento esomico completo di 4 membri
malati della famiglia e il completo perfezionamento della regione al WMS 2012. I dati
sono stati quindi pubblicati: una mutazione nella fase di lettura nel gene della
trasportina 3 (TNPO3) è condivisa da tutti i membri malati della famiglia con un 94% di
penetranza. Il gene TNPO3 è composto da 23 esoni e codifica una proteina di 923
amminoacidi, espressa anche nel muscolo scheletrico. La proteina con la mutazione
nella fase di lettura TNPO3 è più grande di quella del ceppo selvatico, dato che le
manca il suddetto stop codon e si trova attorno al nucleo ma non dentro. I pazienti con
un esordio nell'adolescenza mostrano un fenotipo più severo con una progressione
rapida, mentre i pazienti con un esordio in età adulta presentano una progressione più
lenta. Hanno un’atrofia marcata dei muscoli del cingolo pelvico, coinvolgendo
specialmente il vastus lateralis e l’ileopsoas. È interessante che alcuni pazienti
presentano disfagia, aracnodattilia e insufficienza respiratoria. Il range del CK è di 1–3
volte. Non si è riscontrato coinvolgimento cardiaco.
Ringraziamenti
Questo studio è stato supportato principalmente dai fondi di Telethon, Italia (TGM11Z06 to V.N.) e
Telethon- UILDM (Unione Italiana Lotta alla Distrofia Muscolare) (GUP 10006 and GUP11006 to V.N.). I
finanziatori non hanno avuto nessun ruolo nello studio, raccolta ed analisi dei dati, decisione sulla
pubblicazione o preparazione del manoscritto.
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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 e
genetiche di una numerosa famiglia italo-spagnola con LGMD autosomica dominante,
precedentemente mappata in 7q32.1–32.2 (LGMD1
F).
Abbiamo raccolto le cartelle cliniche in 19 di 60 pazienti; in un paio di malati si è
studiata l’analisi istopatologica delle biopsie muscolari (madre 1 biopsia, sua figlia 2
biopsie consecutive ai 9 e 22 anni). Si è osservato che l’età d’esordio variava dai 2 ai 35
anni e si è verificata tanto nel cingolo pelvico che in quello scapolare. In 14 casi si è
riscontrata ipotrofia tanto nei muscoli prossimali superiori quanto nelle estremità inferiori
nei polpacci. La gravità non è aumentata nelle successive generazioni. Conclusioni
cliniche non precedentemente notificate sono aracnodattilia, disfagia e disartria.
Inoltre, abbiamo riscontrato discrepanza tra la gravità clinica e la biopsia muscolare: la
figlia ha un decorso clinico più grave, nella prima biopsia aveva unicamente atrofia delle
fibre tipo 1, mentre l’atrofia delle fibre è aumentata nella seconda biopsia. La madre
aveva una istopatologia del muscolo più compromessa (più variabilità delle fibre
muscolari e cambiamenti autofagici con macchie di fosfatasi acida). La causa
12
dell’atrofia progressiva e la perdita di miofibrille è un assemblaggio sarcomerico
anormale. Mediante microscopio elettronico si è individuato un accumulo di corpi
miofibrillari nelle fibre muscolari. Si è osservato un accumulo di desmina e miotilina e
aggregati p62.
Si è scoperto come causa di questa malattia un difetto nel gene della trasportina 3 che
rappresenta un nuovo meccanismo di miopatia dominante. I nostri dati morfologici e
ultrastrutturali sembrano seguire un fenotipo simile alle malattie miofibrillari; tuttavia,
erano presenti anche autofagosomi. È possibile che le proteine SR non possano
migrare o essere trasportate fuori e dentro dalla membrana nucleare.
Ot
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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 dominante
che colpisce una famiglia spagnola. Usando un sequenziamento genomico completo, si
è identificata la delezione di un unico nucleotide (c.2771del) nel gene della trasportina 3
in un paziente con LGMD1F. La mutazione interrompe il codone di stop di TNPO3 e
causa una mutazione nella fase di lettura. La trasportina 3 è una proteina nucleare e
media l’importazione delle proteine ricche in serina-arginina al nucleo, che sono
importanti per lo splicing del mRNA. L’oggetto dello studio è l’analisi della trasportina 3
nella patogenesi della LGMD1F.
Si è eseguito un sequenziamento del TNPO3 mediante dideossi in 24 pazienti malati e
23 familiari sani. I campioni muscolari di 4 pazienti sono stati analizzati mediante metodi
convenzionali e immunoistochimica. Un sequenziamento diretto di TNPO3 ha mostrato
che tutti i pazienti avevano una mutazione eterozigote e nessuno dei familiari sani
aveva 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 pazienti
studiati.
La immunistochimica con anticorpi antitrasportina 3 mostrano una colocalizzazione con
i nuclei nei soggetti di controllo. Nei pazienti, si è anche osservata la trasportina 3 nel
nucleo, ma spesso disegualmente distribuita nella periferia, in un pattern di colorazione
a macchie simile a quello osservato con HE. Gli studi genetici e istologici in una famiglia
spagnola sostengono fortemente l’ipotesi che il gene TNPO3 è la causa genetica della
LGMD1F. Gli studi patologici indicano anche che la distribuzione subcellulare della
trasportina 3 è interrotta e colpisce la struttura dei nuclei.
13
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Annalaura Torella1,2, Marina Fanin3, Margherita Mutarelli1, Enrico Peterle3, Francesca
Del Vecchio Blanco2, Rossella Rispoli1,4, Marco Savarese1,2, Arcomaria Garofalo2, Giulio
Piluso2, Lucia Morandi5, Giulia Ricci6, Gabriele Siciliano6, Corrado Angelini3,7, Vincenzo
Nigro1,2
1. TIGEM (Telethon Institute of Genetics and Medicine), Napoli, Italia,
2. Dipartimento di Biochimica Biofisica e Patologia Generale, Seconda Università degli Studi di
Napoli, Napoli, Italia
3. Dipartimento di Neuroscienze, Università degli Studi di Padova, Padova, Italia
4. Cancer Research UK, Londra, Regno Unito
5. Fondazione IRCCS Istituto Neurologico C. Besta, Milano, Italia
6. Dipartimento di Medicina Clinica e Sperimentale, Università degli Studi di Pisa, Pisa, Italia
7. IRCSS S. Camillo, Venezia, Italia
Riassunto
Le Distrofie Muscolari dei Cingoli (LGMD) hanno condizioni genetiche e cliniche
eterogenee. Abbiamo studiato una numerosa famiglia con un pattern di trasmissione
autosomica dominante, precedentemente classificata come LGMD1F e mappato nel
cromosoma 7q32.
I membri malati si caratterizzano per debolezza muscolare che colpisce prima il cingolo
pelvico e l’ileopsoas.
Abbiamo sequenziato l’esoma completo di 4 membri della famiglia ed abbiamo
identificato una variante condivisa della mutazione eterozigote della fase di lettura nel
gene trasportina 3 (TNPO3), che codifica un membro della superfamiglia importina-β. Il
gene TNPO3 è mappato nell’intervallo critico della LGMD1F e il prodotto genico umano
dei suoi 923 amminoacidi è espresso anche nel muscolo scheletrico. Inoltre, abbiamo
identificato un caso isolato di LGMD con una nuova mutazione missenso nello stesso
gene. Abbiamo localizzato il TNPO3 mutante attorno al nucleo, ma non dentro. Il
coinvolgimento genetico connesso con il trasporto al nucleo suggerisce un nuovo
meccanismo patologico che conduce alla distrofia muscolare.
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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, Italia
5. IRCCS Ospedale San Camillo, Venezia, Italia
Riassunto
Delle 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 le
caratteristiche cliniche, istopatologiche e MRI muscolare di una numerosa famiglia italospagnola con LGMD1F che presenta debolezza nei muscoli prossimali degli arti e nei
muscoli assiali. Abbiamo ottenuto dati clinici completi e classificato la progressione della
malattia in 29 pazienti. Si è realizzata una MRI muscolare in 7 pazienti. Si sono studiate
3 biopsie muscolari di 2 pazienti. I malati con un’età di esordio nelle prime decadi
14
presentano un fenotipo più severo con un decorso rapido della malattia, quelli con
esordio da adulti presentano uno sviluppo più lento. La MRI muscolare mostra
un’importante atrofia nei muscoli degli arti inferiori, specialmente nel vastus lateralis.
Ampliare la popolazione dei pazienti ha permesso l’identificazione di caratteristiche
precedentemente non segnalate, includendo disfagia, aracnodattilia e insufficienza
respiratoria. Le biopsie muscolari mostrano atrofia diffusa delle fibre che evolve nel
tempo, cambiamenti miopatici cronici, aree basofile citoplasmatiche, autofagosomi e
aggregati miofibrillari e proteine citoscheletriche. La LGMD1F si caratterizza per un
coinvolgimento selettivo dei muscoli degli arti e insufficienza respiratoria in fase
avanzata e per diversi gradi di progressione clinica. Nuove caratteristiche cliniche sono
emerse dallo studio di ulteriori pazienti.
L’impiego di sequenziatori di ultima generazione (NGS) in questa famiglia ha dato come
risultato la recente identificazione della trasportina 3 (TNPO3) come la causa genetica
della LGMD1F (13). Questi nuovi risultati sono cruciali per capire il nesso tra i
meccanismi patogenetici e le caratteristiche cliniche.
Mar
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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, USA
4. Dipartimento di Patologia e Neuropatologia, Istituto per la Ricerca Biomedica di Vigo (IBIV),
Ospedale Universitario di Vigo (CHUVI), Vigo, Spagna
5. Dipartimento di Neurologia, Ospedale Universitario e Politecnico La Fe, Valencia, Spagna, e
Biomedical Network Research Centre on Neurodegenerative Disorders (CIBERNED), Istituto di
Salute Carlos III, Madrid, Spagna
6. Clinica per le Malattie Neuromuscolari, Dipartimento di Neurologia, Ospedale Universitario Vall
d’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, Spagna
9. 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 di
distrofia muscolare dei cingoli, la distrofia muscolare dei cingoli 1F, nel cromosoma
7q32.1-32.2, ma l’identificazione del gene mutante era sfuggente. Adesso, usando una
strategia di sequenziamento dell’intero genoma, abbiamo identificato la causa della
mutazione della distrofia muscolare dei cingoli 1F, una delezione eterozigote di un
singolo nucleotide (c.2771del) nel codone di stop della Trasportina 3 (TNPO3). Questo
gene si colloca dentro la regione cromosomica connessa alla malattia e codifica una
proteina della membrana nucleare appartenente alla famiglia delle beta importine. Il
TNPO3 trasporta proteine ricche di serina/arginina nel nucleo ed è stato identificato
come un fattore chiave nel processo di importazione dell’HI
Vnel nucleo. La mutazione
genera un'estensione di 15 amminoacidi nel terminale C della proteina, isolato con il
fenotipo 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’espressione
dell’RNA messaggero mutante e le anomalie istologiche dei nuclei e TNPO3 indicano
una funzione alterata del TNPO3. I nostri risultati dimostrano che la mutazione del
15
TNPO3 è la causa della distrofia muscolare dei cingoli 1F, ampliando le nostre
conoscenze sulle basi molecolari delle distrofie muscolari e rinforza l’importanza dei
difetti delle proteine dell’involucro nucleare come causa di miopatie ereditarie.
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Corrado Angelini1, Enrico Peterle2, Marina Fanin3, Giovanna Cenacchi4 and Vincenzo
Nigro5
1.
2.
3.
4.
5.
Università di Neuroscienze di Padova, Italia
Università di Patologia di Bologna, Italia
Università di Patologia Generale di Napoli, Italia
CONTESTO: Le LGMD sono un gruppo eterogeneo di malattie genetiche con
debolezza nei muscoli prossimali degli arti e/o distali. Ad oggi sono conosciute 8 forme
di LGMD autosomiche dominanti. Il fenotipo clinico della LGMD1F è caratterizzato da
una notevole varietà, che va da un esordio precoce, con una progressione rapida e
severa a forme meno aggressive. Le caratteristiche cliniche e morfologiche dei pazienti
con LGMD1F non sono state ancora sufficientemente caratterizzate per suggerire una
specifica eziologia.
METODO: Abbiamo raccolto le cartelle cliniche in 19 di 60 pazienti e abbiamo ampliato
l’albero genealogico; in un paio di malati (madre 1 biopsia, sua figlia 2 biopsie
consecutive 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 4
pazienti è stato studiato con la piattaforma MotorChip CGH array per identificare il gene
responsabile.
RISULTATO: Si è osservato che l’età d’esordio variava dai 2 ai 35 anni; in metà dei casi
si è riscontrata ipotrofia tanto nei muscoli prossimali superiori come nelle estremità
inferiori nei polpacci. Inoltre, abbiamo riscontrato discrepanza tra la gravità clinica e il
coinvolgimento della biopsia muscolare: la figlia (caso di riferimento) ha un decorso
clinico più grave, e una maggiore atrofia delle fibre muscolari, invece la madre aveva
una istopatologia del muscolo più compromessa (più variabilità delle fibre muscolari e
cambiamenti autofagici con macchie di fosfatasi acida). Si è osservato un accumulo di
desmina e miotilina e aggregati p62. Mediante microscopio elettronico si è individuato
un accumulo di corpi miofibrillari nelle fibre muscolari. La MRI muscolare nel paziente di
riferimento mostra una severa e selettiva atrofia nel vastus lateralis.
CONCLUSIONI: I nostri studi morfologici ed ultrastrutturali sembrano suggerire una
miopatia con fenotipo analogo a quelli descritti per malattie Z-disk. Anche se lo specifico
difetto genetico non è ancora noto, è possibile ipotizzare che LGMD1F possa portare ad
una 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 del
Comitato 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.
16
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Giovanna Cenacchi1, Enrico Peterle2, Marina Fanin2, Valentina Papa1, Roberta Salaroli1
and 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 stata
segnalata debolezza muscolare dei muscoli prossimali degli arti e dei muscoli assiali.
Sono state descritte le caratteristiche cliniche, genetiche e istologiche. È stato
precedentemente identificato il locus nel cromosoma 7q32.1-32.2 per questa distrofia
muscolare dei cingoli 1F (LGMD1F). Segnaliamo uno studio patologico muscolare di 2
pazienti (madre e figlia) di questa famiglia. I risultati morfologici muscolari mostrano un
incremento della variabilità della dimensione delle fibre, atrofia delle fibre e vacuoli
positivi alla fosfatasi acida. L’immunofluorescenza per desmina, miotilina, p62 e LC3 ha
mostrato un accumulo di miofibrille, aggregati di proteine leganti l’ubiquitina e
autofagosomi.
Lo studio ultrastrutturale conferma i vacuoli autofagosomali.
Si sono rilevate diverse alterazioni dei componenti miofibrillari, come un importante
disordine, strutture bastoncellari con aspetto granulare e occasionalmente corpi
citoplasmatici. I nostri dati ultrastrutturali e le caratteristiche patologiche muscolari sono
caratteristiche della LGMD1F e sostengono l’ipotesi che i difetti genetici portano a una
miopatia fenotipica associata a un scomposizione della rete citoscheletrica.
I nostri dati morfologici e ultrastrutturali suggeriscono nei nostri casi di LGMD1F una
miopatia fenotipica simile a quelle descritte per le malattie Z-disk. Anche se i difetti
genetici sono ancora in fase di studio, è possibile ipotizzare che la proteina mutante in
LGMD1F possa provocare una scomposizione della rete citoscheletrica desmina
correlata.
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M. Savarese 1, A. Torella 1, M. Mutarelli 2, M. Dionisi 2, T. Giugliano 3, G. Di Fruscio 3, M.
Iacomino 3, A. Garofalo 3, S. Aurino 3, F. Del Vecchio Blanco 3, G. Piluso 3, L. Politano 4,
M. Fanin 5, C. Angelini 5, V. Nigro 3
1. Seconda Università degli Studi di Napoli, Laboratorio di Genetica Medica, Dipartimento di
Patologia Generale, Napoli, 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 nostra
conoscenza dei diversi aspetti della biologia. Può essere inoltre molto potente per
studiare pazienti con condizioni genetiche eterogenee, come le distrofie muscolari. In
primo 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 conoscenza
sull’impatto delle mutazioni nell’espressione e splicing del mRNA nei muscoli malati.
17
Abbiamo utilizzato NGS per identificare nuovi geni attraverso il sequenziamento
dell’intero esoma. Abbiamo sequenziato l’intero esoma di 4 membri della famiglia con
LGMD1F separati da più di undici meiosi ed è stata identificata una singola condivisa
nuova variante eterozigote frame shift. Questo causa un'alterazione nonstop nel gene
della Trasportina 3 (TNPO3) che codifica un membro della superfamiglia delle importine
b. Per raggiungere il secondo compito, abbiamo prima reclutato 160 casi familiari di
distrofia muscolare dei cingoli non specifica con un'apparente eredità autosomica. Tutti i
campioni di DNA sono stati prima arricchiti con 486,480 bp, con una copertura di 2447
esoni di 98 geni usando la tecnologia Haloplex con l’utilizzo di codici a barre. Abbiamo
eseguito aggregati NGS di tutti i campioni e identificato un numero di mutazioni,
verificate poi con sequenziamento Sanger. I casi sono inoltre stati studiati con
piattaforma AgilentMotorChip CGH array versione 3.0 per identificare delezioni o
duplicazioni. Infine, nei casi selezionati, abbiamo eseguito RNA-Seq partendo da un
campione di biopsia muscolare. Abbiamo convertito mRNA in cDNA e lo abbiamo
purificato con un personalizzato SureSelec t Target Enrichment System, focalizzato
sugli stessi 98 mRNAs . Le sonde hanno una copertura 4x con un target totale di 1.41
Mb di sequenze/campioni. Questi cDNA sono stati sequenziati usando codici a barre
cercando di ottenere una copertura media di sequenziamento di 100x. I nostri risultati
confermano che c’è un'eterogeneità molto alta nelle distrofie muscolari e che i test di
DNA e RNA basati su NGS sono pronti per uso diagnostico.
Gi
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2
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C. Angelini, M. Fanin, E. Peterle (Padova, IT)
CONTESTO: Proponiamo nuovi modelli di misurazione quantitativa dell’atrofia
muscolare: 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 15
cm dalla testa del femore (seconda slide della MRI nelle estremità inferiori). In queste
sequenze abbiamo misurato l’area muscolare del quadricipite femorale sinistro e del
vastus lateralis sinistro. Queste misurazioni sono state compiute su 11 pazienti con
diversi tipi di miopatie p.e. due casi di miopatie da accumulo di lipidi, una sclerosi
laterale amiotrofica, 1 distrofia facio scapolo omerale, 1 miopatia miofibrillare, 1
miopatia metabolica, 2 pazienti con LGMD2A, 1 paziente con LGMD1
F, 1 miosite
ossificante, 1 miopatia aspecifica. Le biopsie muscolari di questi pazienti sono state
ulteriormente analizzate con morfometria e marcatori molecolari dell’atrofia o autofagia,
p.e. MURF, LC3.
RISULTATI: Abbiamo eseguito la misurazione dell’area muscolare del quadricipite
femorale (QI) in 11 pazienti, che è risultata in media 3711 mm2 ± SD 792. In questo
gruppo di pazienti abbiamo identificato 2 sottogruppi, uno che include 5 pazienti con un
alto grado di atrofia muscolare (gruppo con alta atrofia) i cui valori sono compresi tra
2400 e 3400 mm2 (media 2966) e uno che include 6 pazienti con un basso grado di
atrofia (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 di
963 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 1400
mm2 (media 1217.8)
CONCLUSIONI: Sia l’indice dei quadricipiti che del vastus lateralis sembra utile per
valutare l’atrofia muscolare nelle LGMD, SLA e miopatie metaboliche: un alto grado di
atrofia del QI è stato trovato nelle calpainopatie, malattie del motoneurone e distrofia
muscolare dei cingoli tipo 1F, la misurazione del VLM è apparsa meno specifica dato
che comprende un'area più vasta. Entrambi questi indici quantitativi ottenuti dalla MRI
muscolare, possono essere usati come risultati clinici della terapia in malattie
18
neuromuscolari in modo da seguire e studiare la storia naturale o gli effetti dei vari tipi di
terapie (steroidi, carnitina, ecc.). Un promettente campo di ricerca appare essere la
correlazione degli indici delle immagini con altri parametri di atrofia ottenuti nelle biopsie
muscolari, p.e. con sezione o fibre o marcatori molecolari dell’atrofia e autofagia.
Ot
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G. Cenacchi, E. Peterle, L. Tarantino, V. Papa, M. Fanin, C. Angelini
Dipartimento clinico delle Scienze Radiologiche e Istopatologiche, Università di Bologna Dipartimento di
Neuroscienze e VIMMM, Università di Padova
In una numerosa famiglia italospagnola con apparente eredità autosomica dominante è
stata segnalata debolezza muscolare dei muscoli prossimali degli arti e dei muscoli
assiali. Sono state descritte le caratteristiche cliniche, genetiche e istologiche in 5/32
pazienti. È stato precedentemente identificato il locus nel cromosoma 7q32.1-32.2, ma
nessun difetto è stato rilevato nella Filamina C, un gene candidato da questa regione
cromosomica che codifica proteine leganti l’actina altamente espresse nel muscolo.
Resocontiamo uno studio clinico-patologico di due pazienti (madre e figlia) della stessa
famiglia spagnola. L’età di esordio è stato nell’adolescenza: un esordio più precoce
nella 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’aumentata
variabilità della dimensione delle fibre, atrofia delle fibre, tessuto connettivo endomisio e
perimisio e vacuoli positivi alla fosfatasi acida. Lo studio ultrastrutturale conferma atrofia
delle fibre, anormali aggregati mitocondriali e vacuoli autofagosomali contenenti detriti
cellulari e immagini di pseudo mielina: non sono state riscontrate inclusioni filamentose
che sono solitamente associate a HIBM (miopatia ereditaria da corpi inclusi). Molte
alterazioni di componenti miofibrillari sono state facilmente rilevate così come un
importante disordine, strutture bastoncellari con aspetto granulare e occasionalmente
corpi citoplasmatici. I nostri dati morfologici sostengono l’ipotesi che altre proteine
codificanti actina come FSCN3 e KIAA0265 della stessa regione critica, possano
rappresentare degli interessanti geni candidati nel meccanismo patogenetico nella
LGMD1F.
Agost
o2003
Una nuov
a di
st
r
of
i
a muscol
ar
e deici
ngol
iaut
osomi
ca domi
nant
e(
LGMD1
F)
mappat
anel7q32.
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 Vall
d’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.
19
RIASSUNTO: Nel 2001, gli autori descrivono le caratteristiche cliniche di una distrofia
muscolare dei cingoli autosomica dominante (LGMD1F) geneticamente distinta.
Esaminando l’intero genoma con più di 400 marcatori microsatelliti, gli autori hanno
identificato 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 proteine
leganti l’actina altamente espresse nel muscolo, era un ovvio gene candidato, tuttavia
gli autori non hanno rilevato nessun difetto nella Filamina C o il suo prodotto proteico.
Le distrofie muscolari dei cingoli (LGMD) comprendono un gruppo eterogeneo di
malattie ereditarie caratterizzate da una progressiva e predominante debolezza dei
muscoli 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 di
un’ampia famiglia spagnola di oltre 5 generazioni con LGMD e apparente eredità
autosomica dominante (AD); 44 di 76 (58%) figli di genitori malati manifestano la
malattia. L’esame clinico di 61 persone ha mostrato una debolezza muscolare
progressiva in 32, colpendo principalmente i muscoli dei cingoli pelvico e scapolare.
L’analisi del linkage genetico molecolare per esaminare i loci cromosomici associati ad
altre forme di LGMD autosomiche dominanti hanno dimostrato che questa parentela ha
una diversa forma genetica di LGMD–AD. Per localizzare il locus cromosomico della
malattia, abbiamo intrapreso uno scan dell’intero genoma usando marcatori
microsatellite.
Agost
o2000
Di
st
r
of
i
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ar
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iaut
osomi
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acon
segnidiant
i
ci
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one
J. Gamez1, MD; C. Navarro3, MD; A.L. Andreu2, MD; J.M. Fernandez4, MD; L.
Palenzuela2, MS; S. Tejeira 3, MS; R. Fernandez–Hojas3, MS; S. Schwartz 2, 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 diverse
sono state identificate, inclusi 5 tipi autosomiche dominanti (LGMD-AD).
OBIETTIVO: Descrivere le caratteristiche cliniche, istologiche e genetiche di una vasta
famiglia con LGMD e apparente eredità autosomica dominante di oltre 5 generazioni.
METODO: gli autori hanno esaminato 61 membri della famiglia; sono state eseguite
biopsie muscolari a 5 pazienti. L’analisi del linkage ha valutato i loci cromosomici
associati ad altre forme di LGMD-AD.
RISULTATI: Un totale di 32 individui hanno debolezza dei cingoli scapolare e pelvico. La
severità sembra peggiorare nelle successive generazioni. I risultati della biopsia
muscolare sono stati non specifici e compatibili con distrofia muscolare. L’analisi del
linkage con i cromosomi 5q31, 1q11-q21, 3p25, 6q23, e 7q, ha dimostrato che questa
malattia non è allelica alle LGMD tipo 1A, 1B, 1C, 1D e 1E.
CONCLUSIONI: questa famiglia ha una forma geneticamente diversa di LGMD-AD. Gli
autori stanno al momento eseguendo uno scan dell’intero genoma per identificare il
locus della malattia.
20
Pubblicazioni scientifiche
21
December 9th, 2014
LETTER TO THE EDITOR
INCOMPLETE PENETRANCE IN
LIMB-GIRDLE MUSCULAR
DYSTROPHY TYPE 1F
We observed that clinical signs and symptoms of the
disease were progressively more likely to manifest with
increasing age (Fig. 1). This indicates that, in LGMD1F,
Limb-girdle muscular dystrophy (LGMD) type 1F (MIM #
608423) is a rare autosomal dominant disorder whose
locus was mapped1,2 and gene identified3,4 by investigating
the same large family. During examination of this family,
we expanded the original pedigree and the clinical features of the disease. It is characterized by a variable degree
of muscle weakness and functional impairment, with onset
of symptoms either before age 15 (juvenile form) or in
the third to fourth decades (adult form).5 The clinical2genetic investigation of this family revealed that, in
some patients, the disease was transmitted through apparently unaffected parents (incomplete penetrance). To
calculate the exact penetrance rate, we examined both the
clinical phenotype and the genotype of 115 family members. The attribution of clinical status (either affected or
unaffected) was based on a neuromuscular examination
performed by the same physician using a standardized
protocol and by a questionnaire that we designed to identify main disease symptoms (i.e., muscle weakness, gait difficulty, and dysphagia). One hundred fifteen individuals
(including the 27 subjects investigated in the original
search for the gene defect in this family3 and 88 new
individuals previously untested) underwent DNA sample
collection (obtained after written consent). The genotype
(either mutant or non-mutant) was defined using a
mutation-specific test [amplification refractory mutation
system2polymerase chain reaction (ARMS-PCR)] and confirmed by DNA sequencing. The mutation segregating in
this family (c.2771delA in TPNO3 gene encoding transportin-33) was identified in 45 of 115 individuals (39%)
(Fig. 1); among 45 mutant cases, 39 (86.7%) were affected
(at a mean age of 47.5 years) and 6 (13.3%) were unaffected. Two unaffected subjects were younger than age 15
years, with a future chance of developing the disease, and
4 were adult “non-penetrant” individuals (at a mean age of
31.5 years). Furthermore, 3 additional unaffected adults
whose DNA was unavailable, were obligate carriers of
the disease based on the pattern of inheritance. Overall,
the penetrance rate was estimated to be 84.7%.
C 2014 Wiley Periodicals, Inc.
V
FIGURE 1. (A) Mutation-specific test (ARMS-PCR) showing
that the wild-type allele generates a 195 bp-sized band (wt) and
that the mutant allele generates a 221-bp band (m). Mutant
patients (m) display a doublet of bands corresponding to the
presence of both heterozygous mutant and wild-type alleles.
(B) Electropherograms showing DNA sequence in a control
(wild-type) and a mutant patient. The position of the single
nucleotide deletion causing a non-stop mutation is indicated by
the arrow. (C) Histogram showing age-dependent penetrance
rate in LGMD1F: the proportion of affected patients among
mutant cases progressively increases with the age of individuals (numbers in parentheses indicate number of individuals in
each age group).
Letter to the Editor
MUSCLE & NERVE
Month 2015
1
22
December 9th, 2014
the penetrance is age-dependent. Incomplete penetrance
may be the effect of modifier genes or may due to
environmental factors. Although the contribution of
epigenetic factors was not explored in this study, we did
investigate the potential role of lifestyle and associated
conditions in determining disease manifestations.
These data show that age-related penetrance is a
characteristic feature of LGMD1F that reduces the predictive value of the genetic test. Because age-related penetrance is a major challenge when attempting to
quantify the genetic risk of a patient’s offspring, our
results may be useful for genetic counseling, especially
in younger patients with the mutation.
Marina Fanin, PhD1
Enrico Peterle, MD1
Chiara Fritegotto, PhD1
Anna C. Nascimbeni, PhD1
Elisabetta Tasca, PhD2
Annalaura Torella, PhD3,4
3,4
1
Department of Neurosciences, University of Padova, Padova,
Italy
2
IRCCS Fondazione San Camillo Hospital, Venice, Italy
3
Department of Biochemistry, Biophysics and General
Pathology, II University of Naples, Naples, Italy
4
Telethon Institute of Genetics and Medicine, Naples, Italy
1. Gamez J, Navarro C, Andreu AL, Fernandez JM, Palenzuela L, Tejeira S,
et al. Autosomal dominant limb-girdle muscular dystrophy: a large
kindred with evidence for anticipation. Neurology 2001;56:450–454.
2. Palenzuela L, Andreu AL, Gamez J, Gamez J, Vila MR, Kunimatsu T,
et al. A novel autosomal dominant limb-girdle muscular dystrophy
(LGMD 1F) maps to 7q32.1-32.2. Neurology 2003;61:404–406.
3. Torella A, Fanin M, Mutarelli M, Peterle E, Del Vecchio Blanco F,
Rispoli R, et al. Next-generation sequencing identifies transportin 3
as the causative gene for LGMD1F. PLoS One 2013;8:1–7.
4. Melia MJ, Kubota A, Ortolano S, Vılchez JJ, Gamez J, Tanji K, et al.
Limb-girdle muscular dystrophy 1F is caused by a microdeletion in
the transportin 3 gene. Brain 2013;136:1508–1517.
5. Peterle E, Fanin M, Semplicini C, Vilchez Padilla JJ, Nigro V,
Angelini C, et al. Clinical phenotype, muscle MRI and muscle pathology of LGMD1F. J Neurol 2013;260:2033–2041.
Published online 00 Month 2014 in Wiley Online Library
(wileyonlinelibrary.com). DOI 10.1002/mus.24539
Vincenzo Nigro, MD, PhD
2
---------------------------------------------------------
Letter to the Editor
MUSCLE & NERVE
Month 2015
23
May 2014
Acta Myologica • 2014; XXXIII: p. 1-12
InvIted revIew
Genetic basis of limb-girdle muscular
dystrophies: the 2014 update
Vincenzo Nigro and Marco Savarese
Dipartimento di Biochimica, Biofisica e Patologia Generale, Seconda Università degli Studi di Napoli and Telethon Institute
of Genetics and Medicine (TIGEM), Naples, Italy
and the respiratory muscles. The clinical course and the
expressivity may be variable, ranging from severe forms
with rapid onset and progression to very mild forms allowing affected people to have fairly normal life spans
and activity levels (1). The term LGMD is becoming descriptive and also comprises clinical pictures of different
diseases. The original definition was given as muscular
dystrophies milder that DMD and inherited as autosomal
traits (2). However, the most severe forms with childhood onset also result in dramatic physical weakness and
a shortened life-span. The advent of next generation sequencing approaches has accelerated the pace of discovery of new LGMD genes. Ten years ago the list included
16 loci (3), while today the LGMD loci so far identified
are thirty-one, eight autosomal dominant and 23 autosomal recessive.
Limb-girdle muscular dystrophies (LGMD) are a highly heterogeneous group of muscle disorders, which first affect the
voluntary muscles of the hip and shoulder areas. The definition
is highly descriptive and less ambiguous by exclusion: non-Xlinked, non-FSH, non-myotonic, non-distal, nonsyndromic, and
non-congenital. At present, the genetic classification is becoming
too complex, since the acronym LGMD has also been used for a
number of other myopathic disorders with overlapping phenotypes. Today, the list of genes to be screened is too large for the
gene-by-gene approach and it is well suited for targeted next generation sequencing (NGS) panels that should include any gene
that has been so far associated with a clinical picture of LGMD.
The present review has the aim of recapitulating the genetic basis of LGMD ordering and of proposing a nomenclature for the
orphan forms. This is useful given the pace of new discoveries.
Thity-one loci have been identified so far, eight autosomal dominant and 23 autosomal recessive. The dominant forms (LGMD1)
are: LGMD1A (myotilin), LGMD1B (lamin A/C), LGMD1C (caveolin 3), LGMD1D (DNAJB6), LGMD1E (desmin), LGMD1F
(transportin 3), LGMD1G (HNRPDL), LGMD1H (chr. 3). The
autosomal recessive forms (LGMD2) are: LGMD2A (calpain
3), LGMD2B (dysferlin), LGMD2C (γ sarcoglycan), LGMD2D
(α sarcoglycan), LGMD2E (β sarcoglycan), LGMD2F (δ sarcoglycan), LGMD2G (telethonin), LGMD2H (TRIM32), LGMD2I
(FKRP), LGMD2J (titin), LGMD2K (POMT1), LGMD2L (anoctamin 5), LGMD2M (fukutin), LGMD2N (POMT2), LGMD2O
(POMTnG1), LGMD2P (dystroglycan), LGMD2Q (plectin), LGMD2R (desmin), LGMD2S (TRAPPC11), LGMD2T (GMPPB),
LGMD2U (ISPD), LGMD2V (Glucosidase, alpha ), LGMD2W
(PINCH2).
Autosomal dominant LGMD
The LGMD1, i.e. the autosomal dominant forms,
have usually an adult-onset and are milder, because affected parents are usually in quite good health at reproductive
age. They are relatively rare representing less than 10%
of all LGMD. Sometimes, they correspond to particular
cases of mutations in genes involved in other disorders,
such as myotilin, lamin A/C or caveolin 3 (Table 1).
Key words: Limb-girdle muscular dystrophies, LGMD, NGS
LGMD1A - LGMD1A may be caused by mutations
in the myotilin (MYOT) gene at chr. 5q31.2. The cDNA
is of 2.2 kb and contains 10 exons. Myotilin is a Z-diskassociated protein. LGMD1A may be considered as an
occasional form of LGMD (4). The first clinical report
was in 1994 (5). The gene was identified in 2000 (6), but
myotilin mutations have been rather associated with myofibrillar myopathy. LGMD1A is characterized by late
Introduction
The term limb-girdle muscular dystrophy refers to a
long list of Mendelian disorders characterized by a progressive deterioration of proximal limb muscles. Very often, other muscles are affected, together with the heart
Address for correspondence: Vincenzo Nigro, via Luigi De Crecchio 7, 80138 Napoli, Italy; Telethon Institute of Genetics and Medicine
(TIGEM), via Pietro Castellino 111, 80131 Napoli, Italy. - E-mail: [email protected]
1
24
May 2014
Table 1. Autosomal dominant limb girdle muscular dystrophy.
Gene
Clinical phenotype
Disease
Locus
Name
Exons
LGMD1A
5q31.2
TTID
10
Protein
(protein function)
Typical
onset
myotilin
(structural; Z disc)
Adulthood
Progression
Cardiomiopathy
sCK
Slow
Not observed
3-4X
Allelic disorders (OMIM, #)
Myopathy, myofibrillar, 3
(609200)
Myopathy, spheroid body
(182920)
Cardiomyopathy, dilated,
1A(115200)
Charcot-Marie-Tooth
disease, type 2B1(605588)
Emery-Dreifuss muscular
dystrophy 2, AD(181350)
Emery-Dreifuss muscular
dystrophy 3, AR(181350)
LGMD1B
1q22
LMNA
12
lamin A/C
(structural; fibrous
nuclear lamina )
Heart-hand syndrome,
Slovenian type(610140)
Variable
(4-38y)
Slow
Frequent
1-6X
Hutchinson-Gilford
progeria(176670)
Lipodystrophy, familial
partial, 2(151660)
Malouf syndrome(212112)
Mandibuloacral
dysplasia(248370)
Muscular dystrophy,
congenital(613205)
Restrictive dermopathy,
lethal(275210)
Cardiomyopathy, familial
hypertrophic(192600)
LGMD1C
3p25.3
CAV3
2
caveolin 3
(scaffolding protein
within caveolar
membranes)
Creatine phosphokinase,
elevated serum(123320)
Childhood
Slow/
moderate
Frequent
10X
Long QT syndrome
9(611818)
Myopathy, distal, Tateyama
type(614321)
Rippling muscle
disease(606072)
LGMD1D
7q36
DNAJB6
10
DnaJ/Hsp40
homolog, subfamily
B, member 6
(chaperone)
Variable
(25-50y)
Slow
Not observed
1-10X
Muscular dystrophy, limbgirdle, type 2R(615325)
LGMD1E
2q35
DES
9
desmin (structural;
intermediate
filament)
1I(604765)
Adulthood
Slow
Frequent
5-10X
Myopathy, myofibrillar,
1(601419)
Scapuloperoneal
syndrome, neurogenic,
Kaeser type(181400)
LGMD1F
7q32
TNPO3
23
transportin 3
(nuclear importin)
Variable
(1-58y)
Slow/
moderate
Not observed
1-3X
-
Variable
(13-53y)
Slow
Not observed
1-9X
-
Variable
(10-50y)
Slow
Not observed
1-10X
-
LGMD1G
4q21
HNRPDL
9
Heterogeneous
nuclear
ribonucleoprotein
D-like protein
(ribonucleoprotein,
RNA-processing
pathways)
LGMD1H
3p23-p25
-
-
-
2
25
May 2014
Genetic basis of limb-girdle muscular dystrophies: the 2014 update
onset proximal weakness with a subsequent distal weakness. Some patients show nasal and dysarthric speech.
Serum CK is normal or mildly elevated. Muscle pathology shows rimmed vacuoles with or without inclusions.
Electron microscopy shows prominent Z-line streaming.
Cardiac and respiratory involvement occasionally occurs.
form. LGMD1D is caused by heterozygous missense
mutations in the DNAJB6 gene at chr. 7q36.3 (10). The
reference cDNA sequence is 2.5kb-long, contains 10 exons and encodes DnaJ homolog, subfamily B, member
6. DNAJ family members are characterized by a highly
conserved amino acid stretch (2) called the ‘J-domain’.
They exemplify a molecular chaperone functioning in
a wide range of cellular events, such as protein folding
and oligomeric protein complex assembly (11). Missense heterozygous mutations of DNAJB6 (p.Phe89Ile,
p.Phe93Leu and p.Pro96Arg) are all located in the Gly/
Phe-rich domain of DNAJB6 leading to insufficient clearance of misfolded proteins. Functional testing in vivo
have shown that the mutations have a dominant toxic effect mediated specifically by the cytoplasmic isoform of
DNAJB6. In vitro studies have demonstrated that the mutations increase the half-life of DNAJB6, extending this
effect to the wild-type protein, and reduce its protective
anti-aggregation effect.
DNAJB6 is located in the Z line and interacts with
BAG3. Mutations in BAG3 are known to cause myofibrillar myopathy (12). A characteristic pathological finding
of LGMD1D is the presence of autophagic vacuoles and
protein aggregation. These protein aggregations contain
DNAJB6 together with its known ligands MLF1 and
HSAP1, and also desmin, αB-crystallin, myotilin, and
filamin C, which are known to aggregate in myofibrillar
myopathy. These results suggest that the phenotype of
LGMD1D also overlaps with that of myofibrillar myopathy.
LGMD1D patients show mildly elevated serum CK
levels. The lower limbs are more affected, particularly the
soleus, adductor magnus, semimembranosus and biceps
femoris. In contrast, the rectus femoralis, gracilis and sartorius and the anterolateral lower leg muscles are mostly
spared. DNAJB6 gene mutations may also be associated
with distal-predominant myopathy. Symptoms in the upper limbs appear later. Some patients develop calf hypertrophy. Onset ranges from 25 to 50 years, with some patients maintaining ambulation throughout life. No cardiac
or respiratory involvement has been reported so far. The
pattern of differential involvement could be identified at
different stages of the disease process.
LGMD1B - LGMD1B is also an occasional LGMD
form caused by lamin A/C (LMNA) gene mutations at chr.
1q22 (7). The reference cDNA is of 3 kb and contains 12
exons. The LMNA gene gives rise to at least three splicing isoforms (lamin A, C, lamin AΔ10). The two main
isoforms, lamin A and C, are constitutive components of
the fibrous nuclear lamina and have different roles, ranging from mechanical nuclear membrane maintenance to
gene regulation. The ‘laminopathies’ comprise different
well-characterized phenotypes, some of which are confined to the skeletal muscles or skin, while others are
multi-systemic, such as lipodystrophy, Charcot-Marie
Tooth disease, progeroid syndromes, dilated cardiomyopathy and Emery-Dreifuss muscular dystrophy (EDMD).
The LGMD1B is characterized by a symmetric proximal
weakness starting from the legs, associated with atrioventricular conduction disturbances and dysrhythmias. CK
is normal to moderately elevated. Most patients develop
proximal leg weakness, followed by cardiac arrhythmias
and dilated cardiomyopathy, with sudden death 20-30
years later. However, there is a continuity between LGMD1B and EDMD (8). Usually the more severe forms of
EDMD with a childhood onset have missense mutations,
whereas the milder LGMD1B is associated with heterozygous truncating mutations: this may arise through a
loss of LMNA function secondary to haploinsufficiency,
whereas dominant-negative or toxic gain-of-function
mechanisms may underlie the EDMD phenotypes.
LGMD1C - LGMD1C is caused by mutations in the
caveolin 3 gene (CAV3) at chr. 3p25.3. The CAV3 gene encodes a 1.4kb mRNA composed of only two exons. Caveolin-3 is a muscle-specific membrane protein and the principal component of caveolae membrane in muscle cells in
vivo: at present this is the only gene in which mutations
cause caveolinopathies (9). LGMD1C is characterized by
an onset usually in the first decade, a mild-to-moderate
proximal muscle weakness, calf hypertrophy, positive
Gower sign, and variable muscle cramps after exercise.
LGMD1E - For the limb girdle muscular dystrophy
originally linked to chr. 6q23 (13) we will use the name
LGMD1E, even if it should be considered, more correctly, as a form of autosomal dominant desminopathy or
myofibrillar myopathy. This form is also known as dilated
cardiomyopathy type 1F (CMD1F). One family previously categorized as having LGMD and dilated cardiomyopathy was reported, indeed, to have the splice site mutation IVS3+3A>G in the desmin (DES) gene at 2q35 (14).
LGMD1D - Autosomal dominant LGMD mapped to
7q36 has been classified as LGMD1E in OMIM, but as
LGMD1D in the Human Gene Nomenclature Committee
Database. In the literature there is another LGMD1D/E
erroneously mapped to 6q, but we will use the acronym
LGMD1D for the 7q-disease and LGMD1E for the 6q-
3
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May 2014
Autosomal recessive LGMD
For desmin see also LGMD2R. As in the desminopathies,
LGMD1E family members show dilated cardiomyopathy
and conduction defects together with progressive proximal muscle weakness starting in the second or third decade. Some family members had a history of sudden death.
Serum creatine kinase is mildly elevated (150-350U/l).
Muscle pathology may show dystrophic changes, but
later the presence of abundant perinuclear or subsarcolemmal granulofilamentous inclusions have been also
observed. The study of these inclusions by laser capture
microdissection followed by mass spectrometry analysis,
led to the identification of the disease-causing mutations
in desmin (14).
The autosomal recessive forms (LGMD2) are
much more common, having a cumulative prevalence
of 1:15,000 (2) with some differences among countries,
depending on the carrier distribution and the degree of
consanguinity.
There are recessive genes in which the loss-of-function
mutations on both alleles tipically result in a LGMD phenotype (ordinary LGMD genes): they correspond to the first
8 forms of LGMD2 (LGMD2A-2H) plus LGMD2L. On
the contrary, other genes (occasional LGMD genes) show a
phenotypic divergence with some mutations associated with
LGMD and other ones determining a more complex disorder. Specific variations in occasional LGMD genes cause the
other forms (LGMD2I-2U). The best examples come from
dystroglycanopathies in which the LGMD presentation is
associated with milder alleles of genes mutated in congenital
forms with brain involvement (Table 2).
LGMD1F - LGMD1F was originally mapped to a
3.68-Mb interval on chromosome 7q32.1-7q32.2 in a
very large Italo-Spanish family. We presented the identification of TNPO3 by whole exome sequencing of four
affected family members and the complete refining of the
region at the WMS 2012. Data were then published (15):
a frame-shift mutation in the transportin 3 (TNPO3) gene
is shared by all affected family members with 94% penetrance. The TNPO3 gene is composed of 23 exons and
encodes a 923-amino acid protein, also expressed in skeletal muscle. The frame-shifted TNPO3 protein is larger
than the wt, since it lacks the predicted stop codon and is
found around the nucleus, but not inside. Patients with an
onset in the early teens, show a more severe phenotype
with a rapid disease course, while adult onset patients
present a slower course. They have a prominent atrophy
of lower limb muscles, involving especially the vastus lateralis and the ileopsoas muscle (16). Interestingly, some
patients present with dysphagia, arachnodactyly and respiratory insufficiency. CK range is 1-3x. No cardiac involvement has been reported.
LGMD2A - LGMD2A is caused by Calpain
3 (CAPN3) gene mutations and represents the most frequent LGMD worldwide (20, 21). The CAPN3 gene spans
53kb of genomic sequence at chromosome 15q15.2 and
the transcript is composed of 24 exons encoding a 94kDa
muscle-specific protein. There is a number of heterozygotes (1:100), carrying many different CAPN3 pathogenic changes. Calpains are intracellular nonlysosomal
cysteine proteases modulated by calcium ions. A typical
calpain is a heterodimer composed of two distinct subunits, one large (> 80 kDa) and the other small (30 kDa).
While only one gene encoding the small subunit has been
demonstrated, there are many genes for the large one.
CAPN3 is similar to ubiquitous Calpain 1 and 2 (m-calpain and micro-calpain), but contains specific insertion
sequences (NS, IS1 and IS2). Calpains cleave target proteins to modify their properties, rather than “break down”
the substrates.
The phenotypic spectrum of calpainopathies is very
broad, but they are true LGMD. For the clinical course,
see also (1).
LGMD1G - LGMD1G has been mapped to chr. 4q21.
Very recently, the defect in the RNA processing protein
HNRPDL has been identified (17) in two different families
by whole exome sequencing. The HNRPDL gene contains
8 exons and is ubiquiously expressed. The gene product is
a heterogeneous ribonucleoprotein family member, which
participates in mRNA biogenesis and metabolism. The
reduced hnrpdl in zebrafish prodeces a myopathic phenotype. Patients show late-onset LGMD associated with progressive fingers and toes flexion limitation (18).
LGMD2B - It is caused by missense or null alleles
of the dysferlin (DYSF) gene (22). The DYSF gene spans
233kb of genomic sequence at chr. 2p13.2 and the major
transcript is composed of 6,911 nt containing 57 exons
in the HGVS recommended cDNA Reference Sequence.
Dysferlin is an ubiquitous 230-KDa transmembrane protein involved in calcium-mediated sarcolemma resealing.
LGMD2B is the second most frequent LGMD2 form (1525%) in numerous countries, but not everywhere (23).
Muscle inflammation is recognized in dysferlinopathy
and dysferlin is expressed in the immune cells.
LGMD1H - By studying a large pedigree from
Southern Italy, a novel LGMD locus has been mapped on
chromosome 3p23-p25.1 (19). Most of patients present
with a slowly progressive proximal muscle weakness, in
both upper and lower limbs, with onset during the fourthfifth decade of life.
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May 2014
Table 2. Autosomal recessive limb girdle muscular dystrophy.
Gene Clinical phenotype Disease
Locus
Name
Exons
Protein product
LGMD2A
15q15
CAPN3
24
Calpain 3
LGMD
Typical onset Progression Cardiomiopathy
phenotype
Moderate/
rapid
sCK
ordinary
Adolescence
Slow
Possible
5-40X
Rapid
Often severe
10–70X
Rarely observed
Allelic disorders
(OMIM, #)
3–20X
Miyoshi muscular
dystrophy 1 (254130)
LGMD2B
2p13.2
DYSF
56
Dysferlin
ordinary
Young
adulthood
LGMD2C
13q12
SGCG
8
γ-Sarcoglycan
ordinary
Early childhood
LGMD2D
17q21.33
SGCA
10
α-Sarcoglycan
ordinary
Early childhood
Rapid
Often severe
10–70X
LGMD2E
4q12
SGCB
6
β-Sarcoglycan
ordinary
Early childhood
Rapid
Often severe
10–70X
LGMD2F
5q33
SGCD
9
δ-Sarcoglycan
ordinary
Early childhood
Rapid
Rarely observed
10–70X
1L (606685)
LGMD2G
17q12
TCAP
2
Telethonin
ordinary
Adolescence
Slow
Possible
10X
1N (607487)
LGMD2H
9q33.1
TRIM32
2
Tripartite motif
containing 32
ordinary
Adulthood
Slow
Not observed
10X
Bardet-Biedl syndrome 11
(209900)
LGMD2I
19q13.3
FKRP
4
Fukutin related protein
ordinary
Late childhood
Moderate
Possible
10-20X
Myopathy, distal, with
anterior tibial onset
(606768)
1G (604145)
Cardiomyopathy, familial
hypertrophic, 9 (613765)
LGMD2J
2q24.3
TTN
312 or
more
Titin
occasional
Young
adulthood
Severe
Not observed
10-40X
Myopathy, early-onset,
with fatal cardiomyopathy
(611705)
Myopathy, proximal, with
early respiratory muscle
involvement (603689)
Tibial muscular dystrophy,
tardive (600334)
Muscular dystrophydystroglycanopathy
(congenital with brain and
eye anomalies), type A, 1
(236670)
LGMD2K
9q34.1
POMT1
20
Protein-O-mannosyl
transferase 1
occasional
Childhood
Slow
Not observed
Muscular dystrophy10-40X dystroglycanopathy
(congenital with mental
retardation), type B, 1
(613155)
Muscular dystrophydystroglycanopathy (limbgirdle), type C, 1 (609308)
LGMD2L 11p13-p12
ANO5
22
Anoctamin 5
ordinary
Variable (young
to late
adulthood)
Slow
Not observed
1-15X
Gnathodiaphyseal
dysplasia (166260)
Miyoshi muscular
dystrophy 3 (613319)
1X (611615)
LGMD2M
9q31
FKTN
11
Fukutin
occasional Early childhood
Moderate
Possible
10-70X
(253800)
(congenital without mental
(613152)
(continues)
5
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May 2014
Table 2. (follows).
Gene Disease
LGMD2N
LGMD2O
Locus
14q24
1p34.1
Name
POMT2
POMGnT1
Clinical phenotype Exons
21
22
Protein product
Protein-O-mannosyl
transferase 2
Protein O-linked
mannose beta1,2-Nacetylglucosaminyl
transferase
LGMD
Typical onset Progression Cardiomiopathy
phenotype
occasional Early childhood
Slow
Rarely observed
sCK
5-15X
Allelic disorders
(OMIM, #)
(613150)
(613156)
(253280)
occasional Late childhood
Moderate
Not observed
2-10X
(613151)
Muscular dystrophydystroglycanopathy (limbgirdle), type C, 3 (613157)
LGMD2P
3p21
DAG1
3
Dystroglycan
singular
Early childhood
Moderate
Not observed
20X
Epidermolysis bullosa
simplex with pyloric atresia
(612138)
LGMD2Q
8q24
PLEC1
32
Plectin
singular
Early childhood
Slow
Not observed
Epidermolysis bullosa
10-50X simplex, Ogna type
(131950)
Muscular dystrophy with
epidermolysis bullosa
simplex (226670)
Muscular dystrophy, limbgirdle, type 2R(615325)
LGMD2R
2q35
DES
9
Desmin (structural;
intermediate filament)
occasional
Young
adulthood
A-V conduction
block
1I(604765)
1X
Myopathy, myofibrillar,
1(601419)
Scapuloperoneal
syndrome, neurogenic,
Kaeser type(181400)
LGMD2S
4q35
TRAPPC11
30
Transport protein
particle complex 11
occasional
Young
adulthood
Slow
Not observed
9-16X
(615350)
LGMD2T
3p21
GMPPB
8
GDP-mannose
pyrophosphorylase B
occasional
Early
childhoodYoung
adulthood
LGMD2U
7p21
ISPD
10
Isoprenoid synthase
domain containing
occasional
Early / Late
Rapid/
Moderate
Possible
6-50X
(614643)
LGMD2V
17q25.3
GAA
20
Alpha-1,4-glucosidase occasional
Variable
Variable
(Rapid to
slow)
Possible
1-20X
Glycogen storage disease
II (232300)
LGMD2W
2q14
LIMS2
7
Lim and senescent cell
antigen-like domains 2
Childhood
-
Possible
-
?
Possible
(615351)
6
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May 2014
named adhalin and contains a “dystroglycan-type” cadherin-like domain that is present in metazoan dystroglycans (35).
LGMD2E - The beta-sarcoglycan gene spans 15kb
of genomic sequence at chromosome 4q11 and the major
transcript is composed of 6 exons. The protein contains of
318 amino acids and weighs 43kDa.
LGMD2F - Delta-sarcoglycan is by far the largest
LGMD gene, spanning 433kb of genomic sequence at
chromosome 5q33.3 and the major transcript is composed
of 9 exons. Intron 2 alone spans 164kb, one the largest
of the human genome. Delta and gamma sarcoglycan are
homologous and of identical size (35kDa).
LGMD2G - Mutations in titin cap (Tcap)/Telethonin
cause LGMD2G, one of the rarest forms of LGMD (36).
Tcap provides links to the N-terminus of titin and other Zdisc proteins. Patients show adolescence-onset weakness
initially affecting the proximal pelvic muscles and then
the distal legs with calf hypertrophy. A homozygous nonsense mutation in the TCAP gene has been described in
patient a congenital muscular dystrophy. The TCAP gene
has also been associated with cardiomyopathy (37), while
common variants may play a role in genetic susceptibility to dilated cardiomyopathy. Immunofluorescence and
Western blot assays may show a Telethonin deficiency.
Full sequencing testing may be cost-effective in all cases,
because the gene is composed only of two small exons.
The telethonin gene (TCAP) spans 1.2kb of genomic
sequence at chromosome 17q12 and the transcript is composed of 2 exons. The protein product is a 19kDa protein
found in striated and cardiac muscle. It binds to the titin
Z1-Z2 domains and is a substrate of titin kinase, interactions thought to be critical for sarcomere assembly.
Only two different mutations have been described in the
TCAP gene in Brazilian patients (36). A mutation (R87Q)
was found in a patient with dilated cardiomyopathy (37).
Moreover, a human muscle LIM protein (MLP) mutation (W4R) associated with dilated cardiomyopathy (DCM) results in a marked defect in Telethonin interaction/localization (38).
LGMD2H - The Tripartite-motif-containing gene
32 (TRIM32) gene spans 14kb of genomic sequence at
chromosome 9q33.1 and the transcript is composed of 2
exons, with the first noncoding and the second encoding
a 673 aa protein of 72kDa. TRIM32 is a ubiquitous E3
ubiquitin ligase that belongs to a protein family comprising at least 70 human members sharing the tripartite motif (TRIM). The TRIM motif includes three zinc-binding
domains, a RING, a B-box type 1 and a B-box type 2, and
a coiled-coil region. The protein localizes to cytoplasmic
bodies. Although the function of TRIM32 is unknown,
analysis of the domain structure of this protein suggests
that it may be an E3-ubiquitin ligase (39).
The “dysferlinopathies” include limb-girdle muscular dystrophy type 2B (LGMD2B) and the allelic forms
Miyoshi myopathy (MM), which is an adult-onset distal form, and distal myopathy with anterior tibialis onset (DMAT), but varied phenotypes are observed. LGMD2B affects earlier the proximal muscles of the arms
whereas MM affects the posterior muscles of the leg.
DYSF gene mutations are associated with heterogeneous clinical pictures ranging from severe functional
disability to mild late-onset forms (24). About 25% of
cases are clinically misdiagnosed as having polymyositis (25). This classification into separate phenotypes does
not reveal true disease differences (26) and the allelic
forms are not due to different mutations. Additional factors (e.g., additional mutations in neuromuscular disease
genes or sport activities that include maximal eccentric
contractions) may worsen the disease expression of causative mutations in dysferlinopathies (27).
WB analysis is very useful and specific (28) when
< 20% level of Dysferlin has been identified, although
Dysferlin can also be increased or secondarily reduced.
NGS-based testing is preferred due to the huge number of
exons to be screened and the lack of mutational hot-spots.
mRNA analysis also works from blood, albeit with some
splice differences (29).
LGMD2C-D-E-F
Loss-of-function mutations in any of the genes encoding the four members of the skeletal muscle sarcoglycan complex, alpha, beta, gamma and delta-sarcoglycan
cause LGMD2D, 2E, 2C and 2F, respectively (30-33).
Sarcoglycans are components of the dystrophin-complex.
They are all N-glycosylated transmembrane proteins with
a short intra-cellular domain, a single transmembrane region and a large extra-cellular domain containing a cluster of conserved cysteines.
Sarcoglycanopathies have a childhood onset, similar
to intermediate form of Duchenne/Becker dystrophies,
and involve both cardiac and respiratory functions. We
consider the possibility to classify these forms apart from
the other LGMD.
LGMD2C - The gamma-sarcoglycan gene spans
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
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May 2014
LGMD2J - TTN is one of the most complex human
genes. The titin gene spans 294,442 bp of genomic sequence at chromosome 2q31 and the major transcript is
composed of 363 exons. It encodes the largest protein of
the human genome composed of 38,138 amino acids with
a physical length of 2 microns. An 11-bp indel mutation
in the last titin exon causes tibial muscular dystrophy and
Gerull et al. (49) showed that a 2-bp insertion in exon
326 of the TTN gene causes autosomal dominant dilated
cardiomyopathy (CMD1G; 604145). A homozygous mutation in the C terminus of titin (FINmaj 11bp deletion/
insertion) causes LGMD2J (50). Titin is the giant sarcomeric protein that forms a continuous filament system in
the myofibrils of striated muscle, with single molecules
spanning from the sarcomeric Z-disc to the M-band (51).
Other “titinopathic” clinical phenotypes are tibial muscular dystrophy (TMD, Udd myopathy) (52) or more severe
cardiac and muscular phenotypes (53).
CAPN3 binds M-band titin at is7 within the region
affected by the LGMD2J mutations and shows a secondary deficiency in the LGMD2J muscle (54). Interactions
with titin may protect CAPN3 from autolytic activation
and removal of the CAPN3 protease reverses the titin
myopathology (55).
The French nonsense mutation (Q33396X) located
in Mex6, seems to cause a milder phenotype than the
typical FINmaj mutation (51). Due to the huge gene size,
NGS sequencing is the only possible way to study this
gene. However, the high number of variants and polymorphisms may have a confounding effect on the diagnosis.
LGMD2K - LGMD2K is caused by hypomorphic
missense mutations in the POMT1 gene at 9q34, containing 20 exons and spanning about 20 kb. Mutations allowing a residual enzyme activity are linked to mild forms.
Different POMT1 alleles, cause congenital muscular
dystrophies due to defects of the dystroglycan glycosylation (MDDGC1) and including severe forms with brain
and eye anomalies or mental retardation (56-58).
LGMD2L - LGMD2L is caused by mutations in the
anoctamin-5 (ANO5) gene at 11p14.3 (59). The ANO5
gene spans 90,192 bp and contains 22 exons; the coding sequence is 2.7kb for 913 amino acids. Alternative
gene names are TMEM16E and GDD1. Anoctamins are a
family of calcium-activated chloride channels (60). This
form of LGMD2 is one of the most frequent in Northern Europe encompassing 10%-20% of cases (61). The
penetrance is probably incomplete, since females are less
frequently affected than males. The most common mutation in Northern Europe is c.191 dupA in exon 5 (62). Patients are usually ambulant and the onset is in adulthood.
They show asymmetric muscle involvement with prevalent quadriceps atrophy and pain following exercise. CK
levels are 5-20x. There is no evidence for contractures,
LGMD2H is usually a late-onset condition characterized by proximal weakness, atrophy, and moderately
raised levels of creatine kinase. Until 2008, the only LGMD2H mutation was Asp487Asn found in Hutterite families (40). Different TRIM32 mutations were then identified in Italian LGMD patients (41) that accounts for about
3% of LGMD2. The D487N mutation of TRIM32 causes
the more severe sarcotubular myopathy (STM). Recently,
two other LGMD2H patients have been described associated with STM morphotype (42).
LGMD2I, LGMD2K, LGMD2M,
LGMD2N, LGMD2O, and LGMD2P
The name dystroglycanopathy has been given to defects due to mutations in six genes (POMT1, POMT2,
POMGnT1, FKTN, FKRP and DAG1) (43). These variations reduce dystroglycan glycosylation and cause a wide
range of phenotypes ranging from mild congenital muscular dystrophies to dramatic conditions, including brain
and eye anomalies (muscle–eye–brain disease or Walker–
Warburg syndrome).
LGMD2I - The fukutin-related protein gene spans
the transcript is composed of 4 exons, with the first three
noncoding. The extracellular part of the dystrophin/
utrophin-associated complex is also involved in congenital muscular dystrophies, as well as in LGMD2I. Fukuyama-type congenital muscular dystrophy (FCMD), is
one of the most common autosomal recessive disorders
in Japan characterized by a congenital muscular dystrophy associated with brain malformation (micropolygria)
due to a defect in the migration of neurons caused by
mutation in the fukutin gene at 9q31 (44). Mutations in
the fukutin-related protein gene (FKRP) at 19q13 cause
a form of congenital muscular dystrophy with secondary
laminin alpha2 deficiency and abnormal glycosylation
of alpha-dystroglycan (45). The same gene is also involved in LGMD2I (15).
All of these diseases are associated with changes in
alpha-dystroglycan expression due to a glycosylation defect of alpha-dystroglycan. Dystroglycan is normally expressed and recognized by polyclonal antibodies, but it is
abnormally glycosylated and not recognized by monoclonal antibodies directed against certain epitopes. FKRP is
resident in the Golgi apparatus. The P448L mutation, that
results in CMD1C, causes a complete mislocalization of
the protein and the alpha-dystroglycan is not processed,
while LGMD2I mutations affect the putative active site
of the protein or cause inefficient Golgi localization (46).
LGMD2I mutations appear to be a relatively common cause of LGMD, accounting for at least 10% of all
LGMD with either severe or mild phenotypes (47, 48).
8
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May 2014
specific transcript Plectin 1f), while there are many other
alternative first exons that are spliced to a common exon
2. These patients produce normal skin plectin and do not
show skin pathology. LGMD2Q patients show early-onset non-progressive or slowly progressive LGMD.
LGMD2R - Desmin is the muscle-specific member
of the intermediate filament (IF) protein family (76). The
desmin (DES) gene at 2q35 contains 9 exons and spans
about 8.4 kb. It encodes a 468-amino acid protein. Autosomal dominant mutations in the DES gene are associated with myofibrillar myopathy (14). The overlap with
the DES gene has also been claimed for LGMD1E (77).
A homozygous splice site mutation has been identified
in two Turkish sibs, born of consanguineous parents, in
intron 7 of the DES gene (c.1289-2A>G), resulting in
the addition of 16 amino acids from residue 428. Since
then, other mutations have been identified. The patients
have onset in their teens or twenties of progressive
proximal muscle weakness and non-specific atrophy affecting both the upper and lower limbs. The serum Ck
is normal. LGMD2R patients usually show A-V conduction blocks but no cardiomyopathy.
LGMD2S - This is caused by mutation in the transport protein particle complex 11 (TRAPPC11) gene that
spans 54,328 bp at chr. 4q35, the mRNA is 4.5kb and
contains 30 exons.
Recently, mutations in TRAPPC11 have been identified in a consanguineous Syrian family with an uncharacterized form of LGMD and in five Hutterite individuals
presenting with myopathy, ID, hyperkinetic movements
and ataxia (78).
TRAPPC11 is a transport protein particle component involved in anterograde membrane transport from
the endoplasmic reticulum (ER) to the ER-to-Golgi intermediate compartment (ERGIC) in mammals (79). Mutations identified so far (c.2938G>A/ p.Gly980Arg and
c.1287+5G>A) cause modifications in TRAPP complex
composition, in Golgi morphology and in cell trafficking. The LGMD2S pathogenic mechanism is similar to
that causing Danon disease, an X-linked myopathy due
to LAMP2 mutations and affecting the secretory pathway (80).
The LGMD2S phenotype ranges from a slowly progressive LGMD with childhood onset and high CK to a
syndrome characterized by myopathy but also neurological involvement (ID and ataxia).
LGMD2T - LGMD2T is caused by milder mutations
in the GDP-mannose pyrophosphorylase B (GMPPB)
gene (81). The GMPPB gene is a small gene of 2,453bp
at chr. 3p21. The mRNA is 1.7kb and contains 8 exons.
Mutations in the GMPPB gene have been associated with
congenital muscular dystrophies with hypoglycosylation
of α-dystroglycan and also with LGMD only in three un-
cardiomyopathy or respiratory involvement. LGMD2L is
allelic with the AD gnathodiaphyseal dysphasia (63) and
with AR distal myopathy (MMD3) (64).
LGMD2M - This is associated with mutations in the
fukutin gene (FKTN) at chr. 9q31.2 (65). The FKTN gene
spans 82,989 bp and contains 10 coding exons, the main
transcript is 7.4kb encoding a protein of 413 amino acids. Also in this case LGMD2M is a milder form caused
by at least one hypomorphic missense mutation in a gene
that, with both non-functional alleles, is associated with
more severe phenotypes (66): WWS, MEB or congenital muscular dystrophies (67). In LGMD2M the CNS is
not affected and the intelligence is normal. Patients are
hypotonic, may be ambulant and the onset is in early
childhood. They show symmetric and diffuse muscle involvement that deteriorates with acute febrile illness. Improvement is seen with steroids. CK levels are 10-50x.
There is also evidence for spinal rigidity, contractures and
cardiomyopathy and respiratory involvement.
LGMD2N - Mutations in the POMT2 gene, containing 21 exons, at chr. 14q24 cause LGMD2N (68). POMT2
is a second O-mannosyltransferase overlapping with
POMT1 expression. POMT2 mutations usually have a
dramatic effect: they cause Walker-Warburg syndrome or
muscle-eye-brain-like (69), but rarely are associated with
LGMD (70). This may occur when the α-dystroglycan
glycosylation is only slightly reduced. In these cases the
mutations are usually missense and the phenotype is characterized by LGMD without brain involvement, very high
serum CK.
LGMD2O - It is associated with milder mutations
in the POMGnT1 gene at chr. 1p32 (71). Usually mutations in the POMGnT1 gene are associated with more
severe phenotypes than LGMD, such as Walker-Warburg
syndrome or MEB. A homozygous hypomorphic allele of
the POMGnT1 gene was found as a 9-bp promotor duplication (72).
LGMD2P - LGMD2P is caused by specific changes
of the dystroglycan (DAG1) gene itself. Recently, Campbell has reported a missense mutation in the dystroglycan
gene in an LGMD patient with cognitive impairment (73).
This substitution interferes with LARGE-dependent
maturation of phosphorylated O-mannosyl glycans on
α-dystroglycan affecting its binding to laminin. As a rule
the dystroglycanopathies are due to mutations in genes
involved in the glycosylation pathway of dystroglycan,
but the dystroglycan gene is normal.
LGMD2Q - This form of LGMD is mutation-specific since other mutations in the Plectin (PLEC1) gene at
chrom. 8q24.3 cause epidermolysis bullosa simplex (74).
LGMD2Q has been identified as a homozygous 9-bp
deletion in consanguineous Turkish families (75). The
deletion affects an AUG that is only present in a muscle-
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May 2014
related patients so far reported. The patients from Indian
and Egyptian descent presented with microcephaly and
intellectual delay. All 3 patients had increased serum creatine kinase and dystrophic findings on muscle biopsy.
Muscle biopsy showed hypoglycosylation of DAG1. The
English LGMD patient was a 6-year-old boy with exercise intolerance and CK = 3,000 UI. Two missense mutations were identified: pAsp27His and p.Val330Ile.
LGMD2U - This is the form caused by some particular alleles of the isoprenoid synthase domain containing (ISPD) gene. The ISPD gene spans 333kb at chromosome 7p21 and contains 10 exons. ISPD mutations
disrupt dystroglycan mannosylation and cause of WalkerWarburg syndrome (82, 83). Mutations in ISPD as well
as TMEM5 genes have been associated with severe cobblestone lissencephaly (84). Null alleles of ISPD produce
Walker Warburg or cobblestone lissencephaly with brain
vascular anomalies, but at least one milder mutation in
one allele has been found in LGMD (68 69). We named
this forms as LGMD2U. The association between mutations in the ISPD gene and LGMD was, however, older
than that of forms 2P-2T, but to avoid discordant definitions among the LGMD2U should be considered as that
caused by some alleles of ISPD. LGMD2U is progressive,
with most cases with LGMD losing ambulation in their
early teenage years, thus following a DMD-like path.
In several patients, there is muscle pseudohypertrophy,
including the tongue. Respiratory and cardiac functions
also decline, resembling other dystroglycanopathies.
LGMD2V - This is a proposal to name as LGMD2V
an occasional LGMD form that derives from mild mutations of the acid alpha-glucosidase (GAA) gene (85). The
GAA gene maps at chr 17q25.3 and comprises 20 exons
with a protein product of 953 aa. Defects in GAA are the
cause of glycogen storage disease type 2 (GSD2, MIM:
232300). GSD2 is a metabolic disorder with a broad
clinical spectrum. The severe infantile form, or Pompe
disease, presents at birth with massive accumulation of
glycogen in muscle, heart and liver. Late-onset Pompe
disease may present from the second to as late as the
seventh decade of life with progressive proximal muscle weakness primarily affecting the lower limbs, as in a
limb-girdle muscular dystrophy. Final outcome depends
on respiratory muscle failure.
LGMD2W - This caused by mutations in the LIM
and senescent cell antigen-like-containing domain protein
2 (LIMS2/ PINCH2) gene at chromosome 2q14. The gene
comprises 7 coding exons. It encodes a 341-aa member
of a small family of focal adhesion proteins. The encoded
protein has five LIM domains, each domain forming two
zinc fingers, which permit interactions which regulate
cell shape and migration. Patients show a childhood onset
LGMD with macroglossia and calf enlargement. They al-
so developed decreased ejection fraction with global left
ventricular dysfunction in their 3rd decade, severe quadriparesis and relative sparing of the face, and characteristically a broad based triangular tongue. This form has been
presented in a poster session at the ASHG 2013.
The classification of LGMD is becoming too complex. We tried to reorganize the different genes so far described following the traditional nomenclature. However
for the autosomal recessive forms there are few letters
available. 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 generating even higher confusion.
Acknowledgements
This study was mainly supported by grants from
Telethon, Italy (TGM11Z06 to V.N.) and TelethonUILDM (Unione Italiana Lotta alla Distrofia Muscolare) (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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Abstracts / Neuromuscular Disorders 23 (2013) 738–852
Hô pital Cochin, Paris,France; 7 AP-HP, Service d’imagerie médicale, Hô
pital Raymond Poincaré, Garches, Garches, France
To determine the clinical characteristics of limb-girdle muscular dystrophy 2E (LGMD2E), and to analyze the genetic and histopathological
features. All LGMD2E patients followed at three European neuromuscular centres were included. The past medical history was collected, and disease 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 populations could be identified according to disease severity: a severe form (n
= 17) with onset <10 years (median 3 years) and early loss of ambula-tion
(13/17pts, median 12 years) and a milder form (n = 10) with later onset
(median 13.5 years) and slower progression (two patients ambulant at 50
and 66 years). Fifty-one mutated alleles were identified (14 muta-tions) in
26 patients; two mutations were recurrent, associated with the severe form
(c.376_383dup, 13/34 alleles) or the milder form (c.-22_10dup32, 8/20). A
hypokinetic or dilated cardiomyopathy was observed in 12 patients (44%,
median 28.5 years). Six patients had a restrictive respiratory insufficiency
requiring ventilation (22%, median 39 years). MRI examinations showed
similar area of fatty replacement more pronounced in the older patient
with longer evolution: Latissimus dorsi, spine extensors and abdominal
belt in trunk; glutei, great and lon-gus adductors in pelvic girdle; anterior
and posterior compartments with sparing of rectus femoris, gracilis,
sartorius and short head of the biceps femoris in thighs. This study refines
the phenotypic spectrum of LGMD2E, and identifies two mutations
predictive of the disease course. The LGMD2E phenotype is associated
with a high incidence of cardiomy-opathy and less frequent respiratory
insufficiency.
http://dx.doi:10.1016/j.nmd.2013.06.459
P.5.8
Why is LGMD2G rare?
C.F. Almeida 1, P.C.G. Onofre-Oliveira 2, M. Zatz 2, L. Negrao 3,
M. Vainzof 2
1
Human Genome Research Center, Institute of Biosciences, University of
Sã o Paulo, Genetics and Evolutionary Biology, Sã o Paulo, Brazil; 2 University of Sã o Paulo, Genetics and Evolutionary Biology, Sã o Paulo, Brazil;
3
Coimbra’s University Hospital, Coimbra, Portugal
Mutations in telethonin gene cause a rare and relatively mild form of
limb-girdle muscular dystrophy type 2G. Only few families were described
presenting this disease, and they are mainly Brazilians. In Brazil, this form
represents less than 5% of all LGMD. In other countries, only isolated
sporadic cases were described in China, Moldavia, Australia and Portugal.
To date, all ten families identified in Brazil present the same c.157C > T
(Q53X) homozygous nonsense mutation. In five families no consanguinity
was referred. All patients also share the same haplotype for microsatellite
markers near the gene, suggesting a common origin of the mutation. Outside Brazil, different mutations were identified in China, Moldavia and
Australian. However, the Portuguese patient described presents the same
mutation found in Brazilians. As a great proportion of Brazilian people
has Portuguese ancestry, and the Brazilian LGMD2G patients show a predominant European genetic background, we consider that the common
mutation arose in Europe and spread through Brazilian population. However, in this case, it would still be expected to find a higher frequency of this
disease in Portugal. By sequencing TCAP exon 2, we found that all the
patients, including the Portuguese one, are homozygous for the allele A of
rs1053651 SNP (the ancestral allele is C). This allele has a frequency of 28%
in the European population. Thus, we hypothesize that the muta-tion may
have occurred in this rarer haplotype, which could explain why
this form is also rare in Portugal. Therefore, the c.157C > T mutation has a
common origin, that implies the occurrence of founder effect, probably in
Portugal and is in linkage disequilibrium with the rs1053651 SNP, which is
compatible with its low frequency in Europe. Financial support: FAPESPCEPID, CNPQ-INCT, FINEP, CAPES-COFECUB.
http://dx.doi:10.1016/j.nmd.2013.06.460
P.5.9
Clinical and molecular analysis of a large cohort of patients with
anoctaminopathy
A. Sarkozy 1, D. Hicks 1, J. Hudson 1, S.H. Laval 1, R. Barresi 2,
M. Guglieri 1, E. Harris 1, V. Straub 1, K. Bushby 1, H. Lochmuller 1
1
Institute of Genetic Medicine, International Centre for Life, Newcastle
upon Tyne, United Kingdom; 2 NSCT Diagnostic & Advisory Service for
Rare Neuromuscular Diseases, Muscle Immunoanalysis Unit, Dental
Hospital, Newcastle upon Tyne, United Kingdom
Recessive mutations in the ANO5 gene cause a spectrum of phenotypes
ranging from isolated hyperCKaemia to limb girdle muscular dystrophy
(LGMD2L), characterized by adult onset proximal lower limb muscular
weakness and raised CK values. The recurrent exon 5 mutation
(c.191dupA) has been found in most of the British and German patients so
far reported. We performed molecular analysis of the ANO5 gene in a large
cohort of undiagnosed patients with clinical suspicion of anoctaminopathy. We identified two pathogenic mutations in 42/205 unrelated
patients (21%), while a single change only was found in further 14 patients.
Fifteen pathogenic changes were novel. The founder c.191dupA mutation
represents 61% of mutated alleles but is confirmed to be less prevalent in
non-Northern European populations. Retrospective clinical analysis of
patients with 2 mutations corroborates previous finding such as the male
predominance and absence of major cardiac or respiratory involvement, as
well as very mild late onset cases of both sexes and isolated hyperCKaemia
only. Our results also confirm anoctaminopathy as one of the most common adult muscular dystrophies in Northern Europe, with a prevalence of
about 20–25% in undiagnosed patients.
http://dx.doi:10.1016/j.nmd.2013.06.461
P.5.10
Clinical and ultrastructural changes in transportinopathy
1
University of Padova, Padova, Italy; 2 University of Bologna, Bologna,
Italy; 3 TIGEM, Napoli, Italy
Muscle histopathological, ultrastructural and genetic features of a
large Italian-Spanish family with autosomal dominant LGMD, previously
mapped to 7q32.1–32.2 (LGMD1F) were studied in 3 biopsies.
We collected the clinical history in 19 of 60 patients; muscle biopsy histopathology was investigated in one pair of affected patients (mother 1
biopsy, her daughter 2 consecutive biopsies at 9 and 22 years).
We observed that the age of onset varied from 2 to 35 years, and
occurred either in upper or in the lower girdle; in 14 cases there was hypotrophy both in proximal upper and in lower extremities in calf muscles.
The severity was not increased in successive generations. Unreported clinical findings were arachnodactyly, dysphagia and dysarthria.
Moreover, we noticed a discrepancy between the clinical severity and
muscle biopsy involvement: the daughter has a more severe clinical course,
the first biopsy had only type 1 fiber atrophy while increased fiber atrophy
was observed in the second biopsy. The mother has a compromised muscle
histopathology (more muscle fiber variation, and autophagic changes by
acid phosphatase stain). An abnormal sarcomeric assembly is the cause
36
October, 2013
767
of progressive atrophy and myofiber loss. Electron microscopy revealed
accumulation of myofibrillar bodies in muscle fibers. Accumulation of desmin and myotilin and p62-positive aggregates was observed.
A defect in transportin-3 gene has been found to be the cause of this
disease, which represents a new mechanism of dominant myopathy.
Our morphological and ultrastructural data seems to suggest a phenotype similar to myofibrillar disease; however, autophagosomes were also
present. It is possible that SR protein cannot migrate or be transported
in- and out-of the nuclear membrane.
Autonoma de Barcelona, Research Group on Neuromuscular and Mitochondrial Disorders, Barcelona, Spain; 3 Institute of Biomedical Research
of Vigo (IBIV), University Hospital of Vigo (CHUVI), Department of
Pathology and Neuropathology, Vigo, Spain; 4 Hospital Universitari i
Politecnic La Fe, Department of Neurology, Valencia, Spain; 5 Hospital
Universitari Vall dHebron, Institut de Recerca, Universitat Autonoma de
Barcelona, Neuromuscular Disorders Clinic,Department of Neurology,
Barcelona, Spain; 6 Columbia University Medical Centre, Department of
Pathology and Cell Biology, New York, United States; 7 Centro Nacional
de Analisis Genomico, Barcelona, Spain
http://dx.doi:10.1016/j.nmd.2013.06.462
Limb-girdle muscular dystrophy 1F (LGMD1F) is an autosomal dominant muscular disease affecting a Spanish family. Using whole genome
sequencing, we identified a single nucleotide deletion (c.2771del) in transportin-3 gene (TNPO3) in a LGMD1F patient. The mutation disrupts the
termination codon of TNPO3 and causes a reading frame shift. Transportin-3 is a nuclear protein, and mediates import of serine–arginine rich proteins into nucleus, which is important for mRNA splicing. This study
aimed to investigate the significance of transportin-3 in the pathogenesis
of LGMD1F.
We performed dideoxy-sequencing of TNPO3 in 24 affected and 23
unaffected family members. Muscle specimens from 4 patients were analyzed by conventional stains and immunohistochemistry. Direct
sequence of TNPO3 revealed that all patients carried a heterozygous
mutation, and none of the unaffected subjects had the mutation. Hematoxylin-eosin (HE) stained muscle revealed nuclei (10.7 ± 3.0%;
mean ± SD) with central pallor in all patients studied. Immunohistochemistry with anti-transportin-3 antibody showed colocalization with
nuclei in control subjects. In patients, transportin-3 was also observed
within nuclei, but was often unevenly distributed in periphery, a staining pattern similar to that seen by HE. Genetic and histological studies
in a Spanish family strongly support the hypothesis that TNPO3 is the
causative gene of LGMD1F. Pathological study also indicates that the
subcellular distribution of transportin-3 is disrupted and affects the
structure of nuclei.
P.5.11
LGMD1D mutations in DNAJB6 disrupt disaggregation of TDP-43
R. Bengoechea 1, E.P. Tuck 1, K.C. Stein 2, S.K. Pittman 1, R.H. Baloh 3,
H.L. True 2, M.B. Harms 1, C.C. Weihl 1
1
Washington University, Neurology, St Louis, United States; 2 Washington
University, Cell Biology and Physiology, St Louis, United States; 3 CedarsSinai Medical Center, Neurology, Los Angeles, United States
Heat shock proteins (HSPs) facilitate the folding or degradation of
misfolded, damaged and aggregated proteins. Disruptions in HSP function may underlie the molecular basis of many degenerative disorders
including some myopathies. The pathogenic mechanism of these chaperonopathies is unclear. We recently identified mutations in DNAJB6, an
HSP40 co-chaperone, as the cause of a hereditary IBM also named
LGMD1D. One feature of LGMD1D muscle is the accumulation of protein inclusions that contain TDP-43. TDP-43 is an RNA binding protein
with a prion-like domain (PrLD) that is mutated in familial amyotrophic
lateral sclerosis (ALS).
LGMD1D mutations in DNAJB6 reside within the highly conserved
G/F domain. Although the role of the G/F domain in DNAJB6 is unclear,
studies in S.cerevisiae, have shown that the homologous G/F domain in
Sis1 (a DNAJB6 ortholog) is required for the propagation of select yeast
prions. Yeast prions contain Q/N rich PrLDs, a feature they share with
TDP-43 and other RNA binding proteins. Consistent with this, homologous LGMD1D mutation in the G/F domain of Sis1 abrogate its ability
to modulate yeast prion propagation.
In mammalian cell culture DNAJB6 associates with TDP-43 in the
nucleus upon heat shock suggesting that TDP-43 is indeed a DNAJB6 client protein. DNAJB6 expression reduces the formation and enhances the
dissolution of TDP-43 positive nuclear bodies. LGMD1D mutant
DNAJB6 expression increases TDP-43 granule formation and slows their
dissolution upon heat shock recovery. This effect is more pronounced in
cells expressing DNAJB6 that lacks the G/F domain. We hypothesize that
LGMD1D mutant DNAJB6 affects localization, aggregation and toxicity
of TDP43. Characterization of a transgenic mouse model of LGMD1D
recently generated in our laboratory will help to elucidate the role of
DNAJB6 and other HSPs in skeletal muscle disease and the complex interplay between RNA binding protein aggregation and disaggregation.
http://dx.doi:10.1016/j.nmd.2013.06.463
P.5.12
A mutation in TNPO3 causes LGMD1F and characteristic nuclear
pathology
A. Kubota 1, M.J. Melia 2, S. Ortolano 3, J.J. Vilchez 4, J. Gamez 5,
K. Tanji 6, E. Bonilla 6, L. Palenzuela 2, I. Fernandez-Cadenas 2,
A. Pristoupilova 7, E. Garcia-Arumi 2, A.L. Andreu 2, C. Navarro 3,
R. Marti 2, M. Hirano 1
1
Columbia University Medical Centre, Department of Neurology, New
York, United States; 2 Vall dHebron Institut de Recerca, Universitat
http://dx.doi:10.1016/j.nmd.2013.06.464
P.5.13
Remarkable muscle pathology in DNAJB6 mutated LGMD1D
S.M. Sandell 1, S. Huovinen 2, J.M. Palmio 1, H. Haapasalo 2, B.A. Udd 1
1
Neuromuscular Research Center, Tampere University Hospital, Neurology, Tampere, Finland; 2 Neuromuscular Research Center, Tampere University Hospital, Pathology, Tampere, Finland
Limb girdle muscular dystrophies are a large group of both dominantly and recessively inherited muscle diseases. Dominantly inherited
LGMD1 diseases are usually milder and later onset forms than recessive
LGMD2. We have followed six Finnish families with LGMD1D and
reported clinical and MRI findings in these families. All families represent
the same DNAJB6 mutation, causing a F93L change in the ubiquitously
expressed co-chaperone DNAJB6. The molecular pathogenesis of
LGMD1D is mediated by defective chaperonal function leading to
impaired handling of misfolded proteins which normally, without the
defect, would be degraded and re-cycled. We have analyzeded 14 muscle
biopsies obtained from 13 patients in six families at very different time
points after onset of muscle weakness symptoms. All biopsies were from
lower limb muscles, either vastus lateralis or gastrocnemius medialis and
processed for routine histology, histochemistry as well as extensive immunohistochemistry and semithin sections with subsequent electron
microscopy.
Uniform findings were myopathic/dystrophic changes in all patients.
Restricted and easily overlooked myofibrillar pathology in routine histopathology included protein aggregates reactive for Z-disk proteins such
37
May 7th, 2013
Distrofia dei cingoli, Telethon scopre il gene responsabile della rara
patologia
07 Maggio 2013
Ricercatori del Tigem di Napoli chiariscono le basi della distrofia dei cingoli di tipo 1F tramite
tecniche di sequenziamento di ultima generazione
Napoli - Identificato il difetto genetico alla base di una rara forma di distrofia muscolare dei cingoli,
quella di tipo 1F: a descriverlo sulle pagine di Plos One è stato un gruppo di ricercatori dell’Istituto
Telethon di genetica e medicina (Tigem) di Napoli, guidati da Vincenzo Nigro, che si sono avvalsi delle
più sofisticate tecnologie di sequenziamento del genoma oggi a disposizione.
"Come suggerisce anche il nome, questa malattia porta a una progressiva debolezza dei muscoli dei
cingoli pelvico e scapolare, compromettendo così la capacità di sollevare pesi e camminare" spiega
Nigro. "Riconoscerla e diagnosticarla correttamente, però, non è facile, perché è molto
eterogenea sia nella sua manifestazione clinica – età di insorgenza e gravità variano molto da un paziente
all’altro – sia dal punto di vista genetico. Ancora oggi, nel 40 per cento dei casi non è possibile
identificare lo specifico gene alterato nel paziente: questo non è velleitario, perché una precisa diagnosi
molecolare innanzitutto conferma il tipo di patologia, poi dà informazioni su come evolverà nel tempo e
permette
di
effettuare
la
consulenza
genetica
agli
altri
componenti
della
famiglia".
Analizzando così il patrimonio genetico di 64 individui di una famiglia italo-spagnola affetti da una
forma di distrofia dei cingoli dalle basi genetiche ancora sconosciute, Nigro e il suo team hanno
identificato il responsabile in un gene localizzato sul cromosoma 7, quello di una proteina chiamata
Transportina 3. I pazienti con questa mutazione presentano, oltre ai segni tipici della distrofia dei cingoli,
debolezza facciale, disfagia, disartria, atrofia e contrattura dei muscoli delle mani, come descritto dai
colleghi dell’Università di Padova guidati da Corrado Angelini. L’analisi genetica è stata possibile grazie
alle apparecchiature all’avanguardia disponibili presso l’Istituto Telethon di Napoli, quelle per il
cosiddetto “next-generation
sequencing”.
"Grazie a questi approcci di straordinaria potenza oggi possiamo analizzare grandi quantitativi di Dna in
tempi relativamente rapidi" continua Nigro. "Basti pensare che lo storico Progetto genoma umano ha
richiesto ben 10 anni e 3 miliardi di dollari per arrivare al sequenziamento del patrimonio genetico
dell’uomo. Oggi con i nostri macchinari possiamo analizzare in soli dieci giorni la parte codificante del
genoma di 48 individui contemporaneamente, per un costo dei reagenti che non supera i 38mila euro. In
pratica, il Dna viene spezzettato, selezionato, sequenziato e poi 'ricomposto' al computer per determinare
la completa sequenza di lettere". Questo lavoro di analisi è molto delicato e richiede alte competenze di
bioinformatica per leggere i dati e trarne delle conclusioni corrette: al Tigem di Napoli ci sono ricercatori
specializzati
proprio
in
questo,
come
Margherita
Mutarelli,
tra
gli
autori
dello
studio.
"Il risultato di questo lavoro è importante innanzitutto per le famiglie, cui possiamo finalmente
fornire una diagnosi molecolare corretta, ma anche per la ricerca: quello messo in luce è un
meccanismo patologico del tutto nuovo, che potrebbe spiegare anche altre malattie simili che colpiscono i
muscoli" conclude Nigro. "Il nostro lavoro, grazie anche al supporto di Telethon, continuerà quindi lungo
due binari: da un lato chiarire il ruolo della proteina che abbiamo identificato come responsabile della
forma 1F di distrofia dei cingoli, dall’altra utilizzare questa stessa tecnologia per andare alla ricerca dei
geni responsabili delle forme ancora “orfane” di questa malattia. Ricordiamoci infatti che anche tra le
malattie rare ce ne sono alcune più trascurate di altre, per le quali cioè non manca soltanto una cura
efficace, ma anche una conoscenza minima di base."
38
May, 2013
Next-Generation Sequencing Identifies Transportin 3 as
the Causative Gene for LGMD1F
Annalaura Torella1,2., Marina Fanin3., Margherita Mutarelli1, Enrico Peterle3, Francesca Del Vecchio
Blanco2, Rossella Rispoli1,4, Marco Savarese1,2, Arcomaria Garofalo2, Giulio Piluso2, Lucia Morandi5,
Giulia Ricci6, Gabriele Siciliano6, Corrado Angelini3,7, Vincenzo Nigro1,2*
1 TIGEM (Telethon Institute of Genetics and Medicine), Napoli, Italy, 2 Dipartimento di Biochimica Biofisica e Patologia Generale, Seconda Università degli Studi di Napoli,
Napoli, Italy, 3 Dipartimento di Neuroscienze, Università degli Studi di Padova, Padova, Italy, 4 Cancer Research UK, London, United Kingdom, 5 Fondazione IRCCS Istituto
Neurologico C. Besta, Milano, Italy, 6 Dipartimento di Medicina clinica e sperimentale, Università degli Studi di Pisa, Pisa, Italy, 7 IRCSS S. Camillo, Venezia, Italy
Abstract
Limb-girdle muscular dystrophies (LGMD) are genetically and clinically heterogeneous conditions. We investigated a large
family with autosomal dominant transmission pattern, previously classified as LGMD1F and mapped to chromosome 7q32.
Affected members are characterized by muscle weakness affecting earlier the pelvic girdle and the ileopsoas muscles. We
sequenced the whole exome of four family members and identified a shared heterozygous frame-shift variant in the
Transportin 3 (TNPO3) gene, encoding a member of the importin-b super-family. The TNPO3 gene is mapped within the
LGMD1F critical interval and its 923-amino acid human gene product is also expressed in skeletal muscle. In addition, we
identified an isolated case of LGMD with a new missense mutation in the same gene. We localized the mutant TNPO3
around the nucleus, but not inside. The involvement of gene related to the nuclear transport suggests a novel disease
mechanism leading to muscular dystrophy.
Citation: Torella A, Fanin M, Mutarelli M, Peterle E, Del Vecchio Blanco F, et al. (2013) Next-Generation Sequencing Identifies Transportin 3 as the Causative Gene
for LGMD1F. PLoS ONE 8(5): e63536. doi:10.1371/journal.pone.0063536
Editor: Paul McNeil, Medical College of Georgia, United States of America
Received February 15, 2013; Accepted March 25, 2013; Published May 7, 2013
Copyright: 2013 Torella et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was mainly supported by grants from Telethon, Italy (TGM11Z06 to V.N. and GTB12001 to C.A.) and Telethon-UILDM (Unione Italiana Lotta
alla Distrofia Muscolare) (GUP 10006 and GUP11006 to V.N.). This work was also supported by grants from the Association Française contre les Myopathies (13859
to M.F. and 14999/16216 to C.A.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]
. These authors contributed equally to this work.
four autosomal dominant LGMD genes are known, encoding
Myotilin (LGMD1A), Lamin A/C (LGMD1B), Caveolin-3
(LGMD1C), and DNAJB6 [7,8] (LGMD1D). Some patients with
mutations in these four genes fulfill the diagnostic criteria for the
LGMDs, but others show a much wider spectrum of different
phenotypes. LGMD1F is a very puzzling disease [9]. It is
characterized by muscle weakness affecting earlier the pelvic
girdle and especially the ileopsoas muscle. Interestingly, some
patients presented with a juvenile-onset form. In the original
article [9], rimmed vacuoles were reported. Recently, immunofluorescence and ultrastructural studies pointed to the presence of
large protein aggregates and autophagosomes [10]. Many
alterations of myofibrillar component were also detected [10].
The critical interval was mapped to a 3.68-Mb interval on
chromosome 7q32.1–7q32.2 [11]. Given the size of the kindred
and the very accurate linkage analysis, the gene identification has
been considered within reach. In this region the obvious candidate
is the FLNC (Filamin C) gene that is mutated in a form of
autosomal dominant myofibrillar myopathy (MFM) with limbgirdle involvement [12], as well as in a second form characterized
by the weakness of distal muscles and non-specific myopathic
features [13]. However, the early onset of some LGMD1F and the
lack of massive protein aggregates of MFM suggest that LGMD1F
may be a different disorder: despite a thorough search, no
mutation was found in the FLNC gene [11]. In addition, other
Introduction
Limb girdle muscular dystrophies (LGMDs) are characterized
by a progressive weakness that begins from the proximal limb
muscles, due to a number of independent genetic defects that are
distinct from the X-linked Duchenne and Becker muscular
dystrophies [1,2]. In addition to the genetic heterogeneity, the
different forms are clinically heterogeneous, with the age at onset
of symptoms varying from early childhood to late adulthood [3].
The milder the symptoms are, more difficult is the LGMD
diagnosis. Magnetic resonance imaging is helpful to characterise
the severity and pattern of muscle involvement [4,5], but
recognition of LGMD type might be hard [6].
Muscle biopsy of LGMD patients generally shows a diffuse
variation in fiber size, necrosis, regeneration and fibrosis, but the
degree of these factors is variable and does not parallel the clinical
severity. Based on the histological features alone, there is scarce, if
any, possibility of diagnosing a specific LGMD form, but western
blot and immunofluorescence can address to the true defect that
can be demonstrated by the finding of a mutation in the
corresponding gene.
The primary distinction is made between the autosomal
dominant (LGMD1) and the autosomal recessive forms (LGMD2),
with an alphabet letter indicating the order of gene mapping [2].
Eight LGMD1 loci have been identified so far. At present, only
PLOS ONE | www.plosone.org
1
May 2013 | Volume 8 | Issue 5 | e63536
39
May, 2013
TNPO3 Is the LGMD1F Gene
(local realignment around in-del and base recalibration) and SNV
and in-del calling were performed with Genome Analysis Toolkit
(GATK) [19].
The called SNV and in-del variants produced with both
platforms were annotated using ANNOVAR [20], the relative
position in genes using RefSeq [21], amino acid change, presence
in dbSNP v137 [22], frequency in NHLBI Exome Variant Server
(http://evs.gs.washington.edu/EVS) and 1000 genomes large
scale projects (http://www.1000genomes.org) [23], conservation
and different prediction algorithms of damaging effect on protein
activity [24,25,26,27] and conservation scores [28,29]. The
annotated results were then imported into an in-house variation
database, also used to make comparisons among samples and filter
results. The alignments at candidate positions were visually
inspected using the Integrative Genomics Viewer (IGV)[30].
The accession number of the dataset of this study is ERP002413
(Sequence Read Archive – EBI at www.ebi.ac.uk).
candidate genes of the region were excluded and LGMD1F
remained unsolved for many years.
In the last few years, the techniques of next-generation
sequencing (NGS) coupled with target enrichment protocols
enhanced the molecular genetic diagnostics [14,15]. We studied
the original Spanish family with additional family members by
exome sequencing [16] using two different NGS platforms. We
sequenced the whole exome of four affected individuals and
identified a number of new variations, one of which was
completely new, shared by all affected subjects, and mapped to
7q32.
Methods
Ethics Statement
This study adhered to the tenets of the Declaration of Helsinki.
Subjects for this study were recruited at Padua University and
exome analysis was performed at the Second University of Naples
and at the Telethon Institute of Genetics and Medicine.
Participants were informed of the nature and risks of the study,
and signed consent forms were obtained. The institutional review
board of the Second University of Napoli (SUN) reviewed and
approved this study (prot. AOP-SUN 862).
Mutation Detection
We designed both cDNA and intronic primers to amplify the
cDNA and the 22 coding exons plus the 3’UTR exon of the
TNPO3 gene (MIM 610032; NM_012470.3, NM_001191028.2)
(Table S2). In addition, we sequenced all the other exons at the
disease interval that were inadequately covered (,10x) (Table S3).
We also designed additional primers to map the alternatively
spliced products. We purified the amplicons and sequenced them
by using the fluorescent dideoxyterminator method on an
automatic sequencer (ABI 3130XL).
Patients
Nineteen patients were included in the clinical study, and they
all fulfilled the diagnostic criteria for LGMD that include a
characteristic pattern of muscular weakness primarily affecting
pelvic girdle, assessed according to MRC Scale and a modified
Gardner-Medwin & Walton scale for proximal LGMD. Age at
onset was assessed as described. We collected blood from 19
patients and 8 healthy relatives. Skeletal muscle biopsy from the
deltoid or vastus lateralis was taken from 2 affected individuals.
Immunoblotting Analysis
For TNPO3 immunoblotting, muscle samples were homogenized in a lyses assay buffer (Urea 8 M, SDS 4%, 125 mM Tris
HCl pH 6.8). The samples were separated on sodium dodecyl
sulphate –9% polyacrylamide gel electrophoresis and transferred
to nitrocellulose membrane. After blocking in 10% no fat dry milk
in Tween-Tris-Buffered Saline (TTBS-1X) buffer (10 mM TrisHCl, 150 mM NaCl, 0.05% TWEEN 20) for 1h, the membranes
were incubated with primary antibodies in TTBS 1X at room
temperature for 2 h. The monoclonal antibody, recognizing a
recombinant fragment (Human) from near the N terminus of
TNPO3, was used in this experiment with a 1:100 dilution
(AbcamH). We also used the rabbit monoclonal antibody AntiTNPO3 antibody [EPR5264] (ab109386) that recognizes a
synthetic peptide corresponding to residues near the C terminal
of Human TNPO3. This was used for WB at 1:300 dilution.
Following primary antibody incubation and rinses, the membranes were incubated with the secondary antibody, goat antimouse immunoglobulin conjugated with horseradish (Sigma), with
1:10,000 dilution in 0.5% dry milk and TTBS 1X. After
45 minutes of antibody incubation and five washes with TTBS
1X buffer, the TNPO3 protein band was visualized with a
chemiluminescence reagent (Supersignal, WestPico, Pierce) and
exposed to X-ray film.
To perform this analysis, Coomassie blue staining was used for
the evaluation of the myosin protein expression to understand the
variations in the levels of the proteins loaded.
Exome sequencing and analysis
Enrichment was performed by hybridization of shotgun
fragment (average size 141 bp) libraries to Agilent SureSelect
Human All Exon 50 Mb (Agilent Technologies, Santa Clara, CA,
USA) in-solution capture assays. Using the SOLiD system v4 (Life
Technologies), we generated an average of 4.2 Gb of mappable
sequence data per sample to achieve ,20x mean coverage of the
targeted exome. The sequences were analyzed using an automated
custom pipeline designed to perform every step of the analysis with
the appropriate program or custom script. Sequencing reads were
first colour-corrected using SOLiD Accuracy Enhancer Tool
(SAET), then mapped to the reference genome (UCSC, hg19
build) using the software BioScope v1.3 (Life Technologies,
Carlsbad, CA, USA) and duplicate reads were removed using
Picard (http://picard.sourceforge.net). Single nucleotide variations
(SNV) and in-del mutation calling analyses were carried out using
the diBayes algorithm with medium stringency settings and the
SOLiD Small Indel Fragment Tool (www3.appliedbiosystems.
com), respectively.
One of the samples was sent to a commercial provider
(Otogenetics Corporation, Norcross, GA, USA) who performed
both whole exome enrichment with the SeqCap EZ Human
Exome Library v2.0 (Roche NimbleGen, Inc, Madison, WI, USA)
and sequencing with the HiSeq2000 platform (Illumina inc., San
Diego, CA, USA). The sequences were analyzed using another
automated pipeline designed to handle Illumina data with custom
scripts and publicly available software. Paired sequencing reads
were aligned to the reference genome (UCSC, hg19 build) using
BWA [17] and post-alignment process and duplicate removal was
performed using SAMtools [18] and Picard. Further processing
Transfection
Plasmid pcDNA6/A encoding N-terminal HA-tagged TNPO3
full length was obtained by NR Landau, NewYork University
School of Medicine [31]. We subcloned (EcoRI-NheI) HA-TNPO3
exons 1 to 17 in pCS2+ and exons 17 to 22 or 23 were amplified
by PCR from cDNA and cloned in pCS2+/HA-TNPO3_1-17
(NheI-XhoI). Four human TNPO3 cDNA constructs were cloned
2
40
May, 2013
Figure 1. LGMD1F family pedigree. Squares represent male; circles represent female; white figures symbolize normal individuals; black figures
indicate individuals with clinical muscular dystrophy. The original LGMD1F family has been extended from subject II,2 and now includes 64 LGMD
patients of both sexes and five non-penetrant carriers (IV-4, V-26, V-29, V-33, and VI-68). The whole-exome sequencing was performed in four patients
indicated by arrows (V-28, VI-36, VI-53, VII-5).
doi:10.1371/journal.pone.0063536.g001
synonymous. Considering the dominant mode of inheritance of
LGMD1F, we focused on the heterozygous calls and discarded all
variants present with a frequency higher than 1% in the NHLBI
Exome Variant Server (http://evs.gs.washington.edu/EVS) or
1000genomes [32] large scale projects. The resulting filtered list of
273 variants was composed of 253 missense, 14 stopgain, 2
frameshift deletions, 2 nonframeshift insertions/deletions and 2
stoploss variations. Only two variants were mapped into the
disease interval between D7S1822 and D7S2519 (positions:
126,287,140-129,964,025) [11]: a nonsynonymous SNV in the
gene IRF5 and a frame-shift deletion that modify the termination
codon in the exon 22 (stoploss) in the TNPO3 on chromosome
7q32.1 at position 128,597,310 (GRCh37/hg19). To verify
whether we could have missed by NGS other shared variants,
we resequenced by the dideoxy-chain termination method all the
coding exons and flanking introns of the full 7q32 region with
lower/absent coverage (Table S3). No other shared unknown
variant was found. In addition, the DNA sample of VI-36 was sent
to a commercial provider for exome sequencing using the Illumina
platform HiSeq2000. Among 153 variations that were shared by
all, the only one in the disease interval was that in the TNPO3
gene (Table 1). Interestingly, this was the only variation of the
whole exome that resulted absent in dbSNP137. We also refined
the interval: the SNP rs45445295 at the SMO gene at position
128,845,555 was present in some affected members (V-8, VI-60,
V-14, VI-11, V-25, V-12), but it was absent in other affected
members (VI-57, VI-27, VI-56) and in all non-affected individuals.
Therefore, the linked region associated with disease locus was
,1.1 Mb smaller (126,287,140-128,845,555) than that reported
by Palenzuela [11].
To confirm the complete co-segregation of the nonstop TNPO3
variant with LGMD1F, we analyzed all available family members,
affected and non-affected. We sequenced by the Sanger method all
the samples and, in addition, we took advantage of an AluI
restriction site that was lost upon mutation. We observed the
into the pCS2HA plasmid : 1) Wt TNPO3 isoform with 22 exons;
2) TNPO3 isoform with 22 exons containing del A p.X924C; 3)
Wt TNPO3 isoform with 23 exons; 4) TNPO3 isoform with 23
exons containing del A p.X924C. We used 500 ng for transient
trasfection of HeLa cells (26105) cells using PolyFect Transfection
Reagent (Qiagen) according to manifacturer’s instruction. Cells
were grown on glass coverslip put into 12 well plates. They were
cultured in Dulbecco’s modified eagle’s medium (DMEM)
supplemented with 10% (v/v) foetal bovine serum and penicillin-streptomycin (GIBCO-Invitrogen) and maintained in a 5%
CO2 incubator at 37uC. 48 hours after transfection, cells were
fixed with 4% paraformaldehyde in PBS for 10 min at RT,
permeabilized in 0,2% Triton X-100 in PBS for 5 min at RT, and
blocked for 1 h in Blocking solution (BSA 6%, Horse Serum 5% in
PBS). Cells were incubated for 1 h at RT with primary antibodies,
followed by 1 h incubation at RT with FITC-conjugated antirabbit and/or Cy3-conjugated anti-mouse antibodies.
Results
Exome analysis
The original LGMD1F family has been extended (Figure 1) to
include additional family members in seven generations starting
from subject II, 2. The updated pedigree includes 64 LGMD
patients of both sexes and five non-penetrant carriers (93%
penetrance). To perform an informative exome sequencing
analysis, we selected four affected family members (VII-5, VI-53,
V-28, and VI-36) with a manifest LGMD phenotype separated by
the largest number of meioses. Interestingly, two family members
(VI-53 and V-28) were absent from the original family used for the
linkage analyses. DNA samples of three individuals (V-28, VI-53,
VII-5) were fragmented, enriched using the SureSelect whole
exome kit and sequenced by SOliD. DNA, muscle RNA and
proteins were extracted for the studies. We found ,20,000 exonic
variations for each sample, 5,722 of which were common to all
three (Table 1 and Table S1) of which 2,471 were non
Table 1. Total and Shared Variants in Patients with LGMD1F.
Patient variant type
V-28
VI-53
VII-5
Shared by all
SOLiD
VI-36
Shared by all four
4,212
exonic/splicing
21,105
21,366
17,123
5,722
17,183
non synonymous
11,852
11,713
9,051
2,471
7,831
1,687
heterozygous
9,348
9,138
6,812
644
4,693
153
frequency in EVS and 1000genomes,1%
6,102
5,785
3,860
273
486
10
Within LGMD1F interval
13
11
5
2
1
1
doi:10.1371/journal.pone.0063536.t001
3
41
May, 2013
Figure 2. Sequence analyses of the TNPO3 mutations. a) Heterozygous delA mutation in Exon 22 of the TNPO3 gene in Proband VII-5. Aligned
electropherograms show mutated (top) and wild-type (bottom) sequences; b) Heterozygous. c.G2453A) in exon 21 of the TNPO3 gene; c) Pedigree of
the isolated case.
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 3. Western blot analysis of skeletal muscle tissue with
antibodies to TNPO3. Equal amounts of muscle proteins from a
LGMD1F patient and a control were run in each lane (10 mg) on a 9%
SDS-polyacrylamide gel and then blotted onto nitrocellulose membrane. In this experiment, we used a monoclonal antibody that
recognizes a recombinant fragment (Human) near the N terminus of
TNPO3 at a 1:100 dilution. A double band is visible in the patient only.
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May, 2013
Figure 4. Indirect immunofluorescence analysis of the wt-hTNPO3 compared with delA p.X924C -hTNPO3. Following transient
transfections, HeLa cells were incubated for 48 h with normal DMEM and detected by anti-HA immunofluorescence. Nuclei are stained with DAPI
(blue). The endogenous protein is recognized using a rabbit monoclonal anti-TNPO3 antibody (green), while the transfected TNPO3 proteins were
HA-tagged (red). a) An accumulation around the nucleus is usually observed using the mutant delA p.X924C -hTNPO3. b) The typical intranuclear
staining pattern can be observed in cells transfected with wt-hTNPO3 (in red) or c) in non transfected HeLa cells.
characteristic LGMD phenotype. Muscular histopathological data
evidenced dystrophic features and, in addition, discrete mitochondrial alterations, with sporadic ragged-red fibers and cytochrome c
oxidase negative fibers. Mutations in the mitochondrial DNA were
excluded.
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Importance of the TNPO3 mutation
nuclear export/import of proteins and in the RNA splicing
mechanism, we have two hypotheses: 1) this mutation blocks the
nuclear export/import because the longer protein is unable to
move to the nucleus, but remains outside the nuclear membrane 2)
the mutated protein does not interact with the cargo proteins,
causing the block of the nuclear import/export.
Our present data indicate that TNPO3 is the gene mutated in
LGMD1F. Additional functional studies in model organisms are,
however, necessary to understand whether the dominant role of
these mutations is due to haploinsufficiency or to a dominantnegative mechanism. This should be possible by the use of
antisense morpholino oligos in Danio rerio (Zebrafish) where a single
and conserved TNPO3 ortholog is present with 792/923 (86%)
amino acid identity (Table S5).
Advances in the knowledge of limb-girdle muscular dystrophies
have been made in the last few years. With LGMD1F, five
different autosomal dominant LGMD genes have been so far
recognized. The use of NGS technologies promises a revolution in
diagnostics and a more rapid characterization of patients.
To analyze the effects of the nonstop mutation on TNPO3 gene
products, we first performed the mRNA analysis using skeletal
muscle biopsy of a patient compared with a normal control. In
both cases, we identified two differently spliced muscular versions
of the gene, both including exon 22. Form A that also join exon 22
to exon 23 that is non coding and form B that ends in exon 22.
These forms encode the same protein, when the DNA sequence is
normal, because the stop codon is in exon 22. However, the
LGMD1F mutation eliminates this stop and, for both forms, the
muscle protein product is extended by the frame-shift. Form A
should be 15 amino acids longer (CSHSCSVPVTQECLF), while
form B should contain additional 95 amino acids.
We then performed immunoblotting analyses of the skeletal
muscle biopsy using the anti TNPO3 antibody. While mutant
form A is virtually overlapping with wild-type form A, a mutant
form B can be appreciated by western blot analysis of muscle
samples as a higher molecular weight band (Figure 3).
We generated a construct expressing the WT and del A
p.X924C allele. HeLa cells were transfected with either the Wt or
the mutant TNPO3. The transfected proteins were distinguished
from the endogenous TNPO3 by adding a HA-tag. Figure 4 shows
that the WT TNPO3 entered the nucleus, while the mutant was
usually around the periphery of the nucleus.
Supporting Information
Table S1
Exome sequencing data.
(DOC)
Table S2
Discussion
Primers designed for TNPO3 amplification and Sanger
sequencing.
(DOC)
Here, we report the identification of a frame-shift variant del A
p.X924C at the TNPO3/Transportin-SR2 gene on chromosome
7q32.1 at position 128,597,310 (GRCh37/hg19) in all patients
with limb girdle muscular dystrophy 1F. No other variant was
shared by four affected members of the family. The variant
modifies the true stop codon and encode for two elongated
proteins of 15 and 95 amino acids. Interestingly, one flanking SNP
(rs12539741 at 128,596,805) has been identified in association
with others in the region as a susceptibility locus for primary
biliary cirrhosis [33]. Considering that the affected family
members may share a ,2.6 Mb-region on chromosome 7q32,
there is the possibility that any other rare heterozygous variant
could co-segregate in cis with the true LGMD1F mutation. Thus,
we searched in a large collection of patients independent TNPO3
disease-associated variations. We found a missense Arg818Pro in
an isolated LGMD case co-segregating with the disease in this
family. This variation was predicted as causative by the nature of
the change and the conservation.
Transportin 3 is a member of the importin b super-family that
imports numerous proteins to the nucleus, including serine/
arginine-rich proteins (SR proteins) that control mRNA splicing
[34,35]. Transportin 1 (TNPO1), also known as karyopherin b-2,
mediates the nuclear import of M9-bearing proteins[36], while
TNPO2 (karyopherin b -2B) participates directly in the export of a
large proportion of cellular mRNAs[37]. There are two main
TNPO3 proteins: variant 1 that is 923 amino acids long and
variant 2 composed of 859 amino acids, while a longer variant of
the 3’ terminus (hTNRSR1[35]) was probably due to a sequence
artifact. The 923-amino acid protein is found in the skeletal
muscle, translated from two equivalent messengers that include or
not the 3’ noncoding exon.
The TNPO3 nonstop allele hindered the nuclear localization of
the protein in HeLa cells. Given the role of TNPO3 protein in the
Table S3
Exons inadequately covered by NGS exome sequencing and primers designed for Sanger sequencing.
(DOC)
Table S4
Co-segregation study.
(DOC)
Table S5
Alignment of Human and Danio r. TNPO3 proteins.
(DOC)
Table S6
Shared SNVs in the four samples sequenced by NGS.
(XLSX)
Acknowledgments
We thank all the patients and their families for their contribution to this
work. We acknowledge the Neuromuscular Bank of Tissues and DNA
samples (NMTB) for collecting samples (C.A. and M.F.), Stefania Crispi
and Luigi Leone at the IGB Facility of Next Generation Sequencing using
the SOLID platform, and Anna Cuomo and Rosalba Erpice for Sanger
sequencing. The authors would like to thank the NHLBI GO Exome
Sequencing Project and its ongoing studies which produced and provided
exome variant calls for comparison: the Lung GO Sequencing Project (HL102923), the WHI Sequencing Project (HL-102924), the Broad GO
Sequencing Project (HL-102925), the Seattle GO Sequencing Project (HL102926) and the Heart GO Sequencing Project (HL-103010). We also
thank Gopuraja Dharmalingam and the TIGEM Bioinformatics Core for
support in exome data analysis and Giuseppina Di Fruscio for the analysis
of isolated cases of LGMD and Marina Mora for helpful suggestions.
Author Contributions
Conceived and designed the experiments: VN MM RR CA. Performed the
experiments: AT AG MF MM EP FDVB MS GP LM VN. Analyzed the
data: CA VN RR MM. Contributed reagents/materials/analysis tools: AT
MF EP GR LM GS CA VN. Wrote the paper: VN.
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DOI 10.1007/s00415-013-6931-1
O RI GIN AL COM MUN ICAT ION
Clinical phenotype, muscle MRI and muscle pathology
of LGMD1F
Enrico Peterle • Marina Fanin • Claudio Semplicini
Juan Jesus Vilchez Padilla • Vincenzo Nigro •
Corrado Angelini
•
Received: 15 March 2013 / Revised: 11 April 2013 / Accepted: 16 April 2013 / Published online: 30 April 2013
Springer-Verlag Berlin Heidelberg 2013
Abstract Of the seven autosomal dominant genetically
distinct forms of LGMD so far described, in only four the
causative gene has been identified (LGMD1A-1D). We
describe clinical, histopathological and muscle MRI
features of a large Italo-Spanish kindred with LGMD1F
presenting proximal-limb and axial muscle weakness. We
obtained complete clinical data and graded the progression
of the disease in 29 patients. Muscle MRI was performed in
seven patients. Three muscle biopsies from two patients
were investigated. Patients with age at onset in the early
teens, had a more severe phenotype with a rapid disease
course; adult onset patients presented a slow course.
Muscle MRI showed prominent atrophy of lower limb
muscles, involving especially the vastus lateralis. Widening the patients population resulted in the identification of
previously unreported features, including dysphagia,
arachnodactyly and respiratory insufficiency. Muscle
biopsies showed diffuse fibre atrophy, which evolved with
time, chronic myopathic changes, basophilic cytoplasmic
E. Peterle M. Fanin C. Semplicini C. Angelini (&)
Department of Neurosciences, University of Padova, Biomedical
Campus ‘‘Pietro d’Abano’’, via Giuseppe Orus 2B,
35129 Padova, Italy
e-mail: [email protected]
J. J. V. Padilla
Servicio de Neurologı́a, Hospital Universitario La Fe,
Valencia, Spain
V. Nigro
Department of Pathology, II University of Naples, Naples, Italy
V. Nigro
Telethon Institute for Genetics and Medicine, Naples, Italy
C. Angelini
IRCCS San Camillo Hospital, Venice, Italy
areas, autophagosomes and accumulation of myofibrillar
and cytoskeletal proteins. The LGMD1F is characterized
by a selective involvement of limb muscles with respiratory impairment in advanced stages, and by different
degrees of clinical progression. Novel clinical features
emerged from the investigation of additional patients.
Keywords Limb girdle muscular dystrophy LGMD1F Clinical phenotype Muscle MRI
Introduction
Autosomal dominant limb girdle muscular dystrophies
(LGMD type 1) are a heterogeneous group of inherited
disorders, which are characterized by progressive
involvement and wasting of proximal limb girdle muscles.
Currently, eight genetically defined autosomal dominant
LGMD subtypes (LGMD1A-1H) have been identified. The
diagnosis of LGMD1 might be obtained on the basis of the
pattern of inheritance, clinical examination, muscle imaging and muscle biopsy. The causative gene has been so far
identified only in four forms, LGMD1A-1D, complicating
the distinction between LGMD1 patients on clinical ground
and promoting a more in-depth knowledge of clinical,
radiological and morphological study. This is a compelling
issue in rare forms of LGMD, such as LGMD1F, which has
previously been reported [1, 2] in the same large Spanish
family with proximal limb and axial muscle weakness we
investigated in the present study. Clinical, histological and
genetic mapping to 7q32.1-2 have been reported in 32
patients, and the anticipation phenomenon was proposed
[1, 2].
The purpose of this paper is to obtain a thorough
investigation of this family with LGMD1F by further
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J Neurol (2013) 260:2033–2041
clinical, radiological and histopathological analysis in an
extended number of patients.
Methods
Patients and neuromuscular examinations
The patients investigated in the present study were
recruited either during an hospitalization or through outpatient examination, organized by a family association.
The clinical data were collected in 24 patients, including
seven previously non-investigated cases, by both a complete clinical neuromuscular examination and a standardized clinical questionnaire, whereas in five cases the data
were obtained only from the clinical questionnaire.
All patients gave informed consent to the participation
to a quantitative neuromuscular evaluation according to the
approved clinical protocol indicated by Local Regulation.
Muscle biopsies and MRI investigation were done after
local ethical committee approval and written consent has
been obtained.
A complete clinical examination was conducted by the
same physician to assess muscle atrophy and hypertrophy,
gait and posture, presence of joint contractures, scoliosis,
scapular winging, individual muscle weakness, difficulty in
climbing stairs or in performing Gowers’ manoeuvre, age
at loss of independent ambulation. Muscle strength of 18
muscle groups, bilaterally, was assessed using the Medical
Research Council (MRC) Scale.
The age at onset and the clinical severity of the disease
at periodical clinical examinations was graded using a
standardized clinical questionnaire which included the
modified Gardner-Medwin and Walton (GM-W) scale:
grade 1 = normal gait, unable to run freely; grade 2 = tiptoe walking, waddling gait, initial Gowers’ sign; grade
3 = overt muscle weakness, climbing stairs with banister;
grade 4 = difficulty rising from a chair; grade 5 = unable
to rise from the floor; grade 6 = unable to climb stairs;
grade 7 = unable to rise from a chair; grade 8 = unable to
walk unassisted; grade 9 = unable to eat, drink or sit
without assistance.
In 24 patients the respiratory function was evaluated by
spirometry in a standing or a sitting position.
Muscle morphometry, histopathology
and immunohistochemistry
We investigated muscle pathology in three biopsies from
two female patients (daughter and mother) who underwent
diagnostic open biopsies (obtained after written consent)
from the left vastus lateralis muscle (case 1, at age 12 and
28 years; case 2, at age 53 years).
Muscle samples were snap-frozen in liquid nitrogenchilled isopentane, cross-sectioned and routinely stained to
assess the histopathological features.
A morphometric study of muscle fibers was conducted
on sections stained for haematoxylin-eosin (H&E), which
were used to digitalize five to seven non-overlapping
random fields, using a 109 microscope objective (Zeiss
Axioskop, Gottingen, Germany). Images were captured
using a Photometrics CoolSnap camera (Roper Scientific,
Ottobrunn, Germany). NIH ImageJ software (v.1.34) was
used to trace the borders of 200–500 fibers and calculate
fiber cross-sectional area (normal range 708–3846 lm2),
fiber diameter (normal range 30–70 lm), coefficient of
size variability (normal range 0–250), and fiber atrophy
factor and hypertrophy factor, which are the expression of
the proportion of abnormally small or large fibers in the
biopsy (normal range 0–150) [3]. These two latter
parameters have been developed to give different importance to fibers with mild or severe degree of change of
fiber size and to detect atrophy or hypertrophy that may
not be otherwise apparent by simply calculating the
average diameter.
Muscle cross sections were processed by immunohistochemistry using a panel of antibodies against desmin
(MAB1698, Chemicon, Temecula, CA, USA; 1:50), myotilin (RSO34, Novocastra Laboratories, Newcastle, UK;
1:100), titin (MCN627, YLEM, Avezzano, Italy; 1:50),
nebulin (N9891, Sigma Chem. St. Louis, MO, USA;
1:50), alpha-actinin (MCV916, YLEM; 1:50), caveolin-3
(610420, BD Transduction Laboratories, Lexington, KY,
USA; 1:50), in order to investigate sarcomeric and myofibrillar and membrane components. For this purpose, 8 lm
thick sections were blocked for 15 min with 1 % bovine
serum albumin in PBS and incubated for 1 h with primary
antibodies. After washes, specific labelling was developed
by immunofluorescence, using anti-mouse cyanine-3 conjugated Ig (Caltag, Burlingame CA) diluted 1:100 and
incubated for 30 min. Sections were mounted with antifading medium and examined with epifluorescence
microscopy.
Immunoblotting of MuRF-1
Conventional immunoblot analysis was conducted using
muscle sections which were dissolved in Laemmli loading
buffer, boiled for 5 min and centrifuged. Proteins were
resolved by SDS-PAGE electrophoresis and blotted to
nitrocellulose membrane. Blots were air-dried, blocked
with 5 % non-fat milk in Tris-Tween-20 saline buffer
(TTBS) and incubated overnight with a polyclonal antibody against MuRF-1 (MP3401, ECM Biosciences,
Versailles, KY, USA), diluted 1:500 in TTBS. After a
thorough washing, the immunoreactive bands were
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2035
visualized using anti-rabbit peroxidase-conjugated antibodies and the chemioluminescent method (GE Healthcare, UK).
The quantity of MuRF-1 protein in the patients’ samples
was determined by densitometry using ImageJ software
v.1.34 (normalizing the MuRF-1 band on blots to the
myosin band in the post-transfer Coomassie blue-stained
gels), and was expressed as a percentage of controls.
MRI imaging
Muscle MRI was performed in seven patients (1 in Padova,
6 in Valencia). A 1.5-T MRI system (Avanto, Siemens,
Erlangen, Germany) was used to investigate body segments
with axial scans in T1-weighted and turbo inversion
recovery magnitude (TIRM) sequences. Patients underwent
scans of the scapular girdle, right upper arm and both lower
limbs. Fibro-fatty replacement was assessed in T1 spinecho sequences. Areas of signal hyper-intensity were
scored as areas of fibro-fatty infiltration, whereas areas of
signal hypo-intensity were interpreted as oedema-like
changes.
The severity of fibro-fatty replacement and its distribution in muscles were scored using the modified Mercuri’s
Scale [4].
We used T1 sequences at the thigh level, at about 15 cm
from the head of the femur, corresponding to the second
slide of MRI in lower extremities, to measure the muscle
area of the left quadriceps femoris and vastus lateralis in 1
LGMD1F and ten patients with various neuromuscular
diseases, matched by sex and age. The borders of the
muscles were outlined on digital MRI images, the area was
calculated using MedStation software (v. 4.9) and expressed as mm2.
Results
Pattern of inheritance
We studied a large LGMD1F family of Italian-Spanish
origin. In our study, the family pedigree has been reconstructed up to the 7th generation, updating to the most
recent generation, and including a novel branch of the
family. The pedigree includes now 61 patients (30 females,
31 males) who are clinically affected with LGMD. The
pattern of inheritance is clearly autosomal dominant, with
high penetrance (94 %), due to some individuals who were
reported to be unaffected even if they transmitted the
mutant allele to their offspring. The anticipation phenomenon, which has previously been suggested [1], was not
confirmed in our series of patients.
Clinical features
We collected the clinical data from a total of 29 patients
(Table 1; Fig. 1).
At onset, the symptoms included difficulty in running or
in climbing stairs and weakness and atrophy in the proximal lower limb muscles. The age at onset ranged from 1 to
31 years (mean = 10.2 ± 6.7). Only one patient had onset
before 5 years and three after age 20 years. At the time of
the clinical study, the patients were aged from 15 to
78 years (mean = 45), and presented a variable degree of
impairment of pelvic girdle muscles, with trouble climbing
stairs or getting up from the floor. In the more advanced
stages of the disease, the weakness involved also the axial
and upper girdle muscles, leading to skeletal deformities,
such as scapular winging, scoliosis, and joint contractures.
One case had drop-head syndrome.
A generalized atrophy of muscle mass was a common
feature, but the muscles more frequently involved were
deltoid and triceps brachii in the upper limbs (Fig. 1), and
the quadriceps femoris and the anterior compartment of the
leg muscles. Specific clinical pointers and indicators for
LGMD1F are skeletal abnormalities such as arachnodactyly (Fig. 1), pes cavus, and mild Achilles tendon retraction. Macroglossia, mild facial weakness, calf hypertrophy
gynecomastia and dysarthria were only occasionally
observed. Dysphagia was found in 8/29 cases and appeared
to be a relatively frequent and previously undescribed
clinical feature.
Table 1 Clinical features in LGMD1F
Characteristics
Number of patients
Infancy onset \5 years
1/29
Childhood/Juvenile onset \15 years
25/29
Adult onset [20 years
3/29
Early loss ambulation \35 years
3/29
Scapular winging
4/29
Calf hypertrophy
2/29
Respiratory involvement
9/24
Scoliosis
13/24
Arachnodactyly
5/24
Achilles tendon contractures
4/29
Pes Cavus
2/24
Gynecomastia
3/24
Macroglossia
1/24
Dysarthria
1/24
Dysphagia
8/29
Mild facial weakness
1/24
In 24 patients a complete clinical evaluation has been obtained; in five
additional patients the clinical data have been obtained by a clinical
questionnaire
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Fig. 1 Pictures from five
different LGMD1F patients.
Note atrophy of upper girdle
muscles, especially deltoid and
triceps brachii (a–e), causing
difficulty in lifting arms over the
head (b) and scoliosis (d). Some
patients showed arachnodactyly
(b, f), finger contractures
(f, h, i) and atrophy of hand
muscles (f, g)
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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 progressive, resulting in three patients in early loss of ambulation (grade 8) before age 30 years. Clinical examination
of such patients revealed an advanced wasting of lower
girdle muscles associated with a severe involvement of the
upper girdle muscles, joint contractures, and severe
impairment of respiratory function. A rapid worsening of
symptoms was reported to have occurred following intense
physical exercise (five cases), alcohol intake (one case),
long periods of inactivity (two cases), benzodiazepine
overuse (one case), and pregnancy (three cases). Another
group of patients presented a more slowly progressing
course of the disease, leading to loss of ambulation after
age 65 years in three cases (Fig. 2).
Nine patients (38 %) had moderate/severe respiratory
involvement (forced vital capacity below 60 % of normal)
that caused also sleep disturbances. Two patients underwent a complete cardiological evaluation (including ECG
and echocardiography) that resulted within normal limits,
and; therefore, cardiological examination was not pursued
in additional cases. Electromyography performed in seven
patients showed myopathic changes. Creatine kinase (CK)
levels measured in seven cases was either normal or up to
threefold increased.
Muscle MRI
According to the T1 sequences, the muscle atrophy that
resulted was more pronounced in the lower limb muscles
than in the upper girdle, affecting mainly the vastus lateralis muscle in the thigh and the triceps surae muscle in the
leg (Fig. 3). The fibro-fatty replacement correlated with the
degree of muscle atrophy, as observed in other forms of
LGMD, i.e., calpainopathy.
In case 1, the area of quadriceps femoris and vastus
lateralis muscles were 62 and 70 % lower (2,395 and
690 mm2, respectively) than the mean of ten neuromuscular controls (3,843 and 991 mm2, respectively).
Muscle histopathology, morphometry,
immunohistochemistry, immunoblotting
Muscle biopsies obtained from the two patients investigated, showed heterogeneous histopathological features
(Fig. 4). Both muscles from case 1 (at age 12 and 28 years)
showed a diffuse and progressive muscle fibers atrophy,
whereas the muscle from case 2 showed chronic myopathic
changes, such as increased fiber size variability, increased
central nuclei, nuclear clumps, fiber splitting, endomysial
fibrosis, type 1 fibers prevalence. Common features of all
three muscle biopsies were basophilic cytoplasmic regions
and increased cytoplasmic reaction for lyososomal acid
phosphatase even in nondegenerating fibers.
In case 2, muscle fiber morphometric analysis (Fig. 5)
revealed normal value of fiber diameters but fiber size
variability was highly increased because of the presence of
Fig. 2 Lineplot describing the
clinical course in 29 LGMD1F
patients. The clinical functional
grade was assessed using the
modified Gardner-Medwin &
Walton scale. Patients showing
a rapid course (wheel-chairbound before age 30 years,
grade = 8) are indicated in
thick line; patients showing a
slower course are indicated in
thin line
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J Neurol (2013) 260:2033–2041
Fig. 3 Muscle MRI T1
sequences from two different
patients at the level of the thigh
(a, c) and calf (b). Note fibrofatty replacement and atrophy of
vastus lateralis in the thigh
(a, c) and triceps surae in the
leg (b)
atrophic and hypertrophic fibers (increased coefficient size
variability, cross sectional area, atrophy factor, and
hypertrophy factor). Conversely, in case 1, the most relevant change was a generalized fiber atrophy (diameter,
atrophy factor, coefficient size variability), which was
significantly increased in the second biopsy done at a distance of 13 years, as compared with the first biopsy
(p \ 0.001) and with the values in case 2 (p \ 0.001).
In both patients, many fibers showed large intracytoplasmic areas with accumulation of cytoskeletal (desmin)
or myofibrillar (myotilin, telethonin) proteins (Fig. 4), and
large protein aggregates.
Immunoblot analysis of MuRF-1 protein, a marker of
ubiquitin–proteasome degradation pathway leading to
muscle atrophy, showed normal expression level in case 2
(98 % of control mean) but highly increased levels in the
second biopsy from case 1 (250 % of control mean) (Fig. 5).
Discussion
The investigation of a previously reported family with
LGMD1F has been expanded by inclusion of additional
seven unreported patients, the clinical follow-up of 17
patients, MRI investigation and further muscle histopathological analysis.
The widening of the patients population in such a rare
form of LGMD, has resulted in the identification of novel
clinical features of the disease. In particular, dysphagia was
observed in 27 % of cases, arachnodactyly with or without
finger contractures was found in 21 % of patients, and
dysarthria and calf hypertrophy were occasionally found.
Typically, the first symptom was difficulty in climbing
stairs. The disease course appeared to be slow and relatively benign in most adult patients. Only three patients
have lost ambulation before age 30 years. Muscle
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J Neurol (2013) 260:2033–2041
2039
Fig. 4 Muscle biopsy from case 1 (28 years) (a–h), and case 2
(54 years) (i–p) stained for H&E (a, b, i), Gomori trichrome (c, j) and
acid phosphatase (d, k, l), and immunolabeled with antibodies against
desmin (e, f, g, m, n), myotilin (h, o) and caveolin-3 (p). A significant
and generalized reduction of fiber size was observed in case 1 (a),
whereas in case 2 there were more prominent chronic changes, such
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)
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30 April 2013
2040
Fiber diameter
A
100
*
*
*
14000
*
*
12000
10000
µ m2
µm
Fiber cross sectional area
*
75
50
8000
6000
4000
25
2000
0
0
Fiber hypertrophy factor
Fiber atrophy factor
1000
1000
750
750
units
units
Fig. 5 Panel A Histograms
showing the mean values of
different muscle fiber
morphometric parameters (Fiber
diameter, Fiber cross sectional
area, Fiber atrophy factor, Fiber
hypertrophy factor, Coefficient
of fibers size variability)
observed in the three muscle
biopsies from two patients
obtained at different ages.
Dotted rectangles indicate the
range of normal values. In case
1, average fiber diameters were
normal but their variability was
highly increased because of
atrophic and hypertrophic fibers.
Conversely, in case 2, the most
relevant change was a
generalized fiber atrophy, which
was significantly increased in
the second biopsy (*p \ 0.001).
Panel B Immunoblot analysis of
MuRF-1 protein in muscle
biopsies from controls (Cntr1,
Cntr2), and case 1 (28 years)
and case 2. After normalization
with myosin protein content in
the post-transfer Coomassiestained gel, MuRF-1 protein
quantity was normal in case 1
(98 % of control mean) and
highly increased in case 2
(250 % of control mean)
J Neurol (2013) 260:2033–2041
500
500
250
250
0
0
Case 1 (12 years)
Coefficient fiber size variability
Case 1 (28 years)
800
Case 2 (54 years)
units
600
B
400
Case
1
Case
2
Cntr
1
Cntr
2
200
0
involvement occurred mostly in the proximal muscles, but
axial muscles were also involved (one case had drop-head
syndrome) and clinical features included dysphagia and
occasional muscle pain.
As compared with LGMD1G [5], that was described in a
family with limb weakness associated with a striking limitation of finger and toe flexors (to be considered as
important clinical indicators), in this family we frequently
observed arachnodactyly and dysphagia.The LGMD1A has
also dysarthria, while LGMD1B is characterized by cardiac
conduction defects [6, 7], which are absent in LGMD1F.
In LGMD1F, muscle MRI demonstrated a proximal
involvement of scapular and pelvic girdles. We used a new
muscle-imaging quantitative index of the area of quadriceps femoris and vastus lateralis muscles, which, even in
the early onset patients, showed a correlation with the
degree of muscle fibre atrophy in the same biopsied
muscle.
The typical clinical symptoms of LGMD1F are progressive muscle atrophy, myopathic EMG, fibro-fatty
replacement of muscle on MRI, and active changes in
muscle biopsy. The cytoplasmic inclusions and myofibrillar desmin-myotilin positive aggregates in muscle fibers
share similarities with the morphological findings observed
in myofibrillar myopathies. The changes we observed in
biopsies from the quadriceps femoris muscles correspond
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J Neurol (2013) 260:2033–2041
2041
to the different degree of involvement observed by muscle
imaging.
Muscle imaging with MRI is increasingly used to
determine the patterns of muscle involvement in LGMD.
The most consistent example is LGMD2A in which a
selective involvement of hip extensors and adductors
muscles is observed. In LGMD2J, tibial muscle involvement is frequently found.
The muscles from our patients showed protein aggregates and autophagosomes [8], analogous to those seen in
other dominant myopathies with protein aggregates, such
as myofibrillar myopathies, and also observed in LGMD1D
and LGMD1A [6, 9–11]. On the contrary, LGMD1H is
characterized by mitochondrial abnormalities in muscle
biopsies with ragged-red fibers [12]. Only Gamez et al. [1]
reported similar mitochondrial changes in LGMD1F. It is
possible that the primary pathogenetic mechanism causes
protein aggregation in the cytoplasm and in the nucleus.
However, to clarify this hypothesis, further experimental
and clinical data are needed. Post-mitotic differentiated
skeletal muscle might be uniquely prone to present toxic
proteins in an aggregated state. In agreement with these
considerations, p62 protein aggregates [8] and MuRF-1
over-protein expression may suggest an involvement of
ubiquitin–proteasome and autophagic degradation pathways in this disorder.
The adoption of the next-generation sequencing (NGS)
strategy in this family resulted in the recent identification
of Transportin-3 (TPNO3) as the causative gene of
LGMD1F [13]. This novel result is crucial to understand
the link between pathogenetic mechanism and clinical
features.
Acknowledgments The authors wish to thank all the family members who promoted meetings for neuromuscular examination and
blood sample collection. This work was supported by grants from the
Association Française contre les Myopathies (13859 to MF, 14999
and 16216 to CA) and the Telethon Italy (GTB12001 and GUP10006
to CA and GUP11006 to UN).
References
1. Gamez J, Navarro C, Andreu AL et al (2001) Autosomal dominant limb-girdle muscular dystrophy: a large kindred with evidence for anticipation. Neurology 56:450–454
2. Palenzuela L, Andreu AL, Gamez J et al (2003) A novel autosomal dominant limb-girdle muscular dystrophy (LGMD 1F)
maps to 7q32.1-32.2. Neurology 61:404–406
3. Dubowitz V, Sewry CA (2007) In. Muscle biopsy: a practical
approach , 3rd edn. Saunders Elsevier, Philadelphia
4. Stramare R, Beltrame V, Dal Borgo R et al (2010) MRI in the
assessment of muscular pathology: a comparison between limbgirdle muscular dystrophies, hyaline body myopathies and myotonic dystrophies. Radiol Med 115:585–599
5. Starling A, Kok F, Passos-Bueno MR, Vainzof M, Zatz M (2004)
A new form of autosomal dominant limb-girdle muscular dystrophy (LGMD1G) with progressive fingers and toes flexion
limitation maps to chromosome 4p21. Eur J Hum Genet
12:1033–1040
6. Hauser MA, Conde CB, Kowaljow V et al (2002) Myotilin
mutation found in second pedigree with LGMD1A. Am J Hum
Genet 71:1428–1432
7. Van der Kooi AJ, van Meegen M, Ledderhof TM, McNally EM,
de Visser M, Bolhuis PA (1997) Genetic localization of a newly
recognized autosomal dominant limb-girdle muscular dystrophy
with cardiac involvement (LGMD1B) to chromosome 1q11-21.
Am J Hum Genet 60:891–895
8. Cenacchi G, Peterle E, Fanin M, Papa V, Salaroli R, Angelini C
(2013) Ultrastructural changes in LGMD1F. Neuropathology.
doi:10.1111/neup.12003
9. Harms MB, Sommerville RB, Allred P et al (2012) Exome
sequencing eveals DNAJB6 mutations in dominantly-inherited
myopathy. Ann Neurol 71:407–416
10. Sarparanta J, Jonson PH, Golzio C et al (2012) Mutations
affecting the cytoplasmic functions of the co-chaperone DNAJB6
cause limb-girdle muscular dystrophy. Nat Genet 44:450–455
11. Hackman P, Sandell S, Sarparanta J et al (2011) Four new Finnish
families with LGMD1D; refinement of the clinical phenotype and
the linked 7q36 locus. Neuromusc Disord 21:338–344
12. Bisceglia L, Zoccolella S, Torraco A et al (2010) A new locus on
3p23-p25 for an autosomal-dominant limb-girdle muscular dystrophy, LGMD1H. Eur J Hum Genet 18:636–641
13. Torella A, Fanin M, Mutarelli M, et al (2013) Next-generation
sequencing identifies Transportin 3 as the causative gene for
LGMD1F. PLoS One (in press)
Conflicts of interest The authors declare that they have no conflict
of interest.
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doi:10.1093/brain/awt074
Brain 2013: 136; 1508–1517
| 1508
BRAIN
A JOURNAL OF NEUROLOGY
Limb-girdle muscular dystrophy 1F is caused by a
microdeletion in the transportin 3 gene
1 Research Group on Neuromuscular and Mitochondrial Disorders, Vall d’Hebron Institut de Recerca, Universitat Autònoma de Barcelona, Barcelona,
08035, Spain
2 Biomedical Network Research Centre on Rare Diseases (CIBERER), Instituto de Salud Carlos III, Madrid, 28029, Spain
3 Department of Neurology, Columbia University Medical Centre, New York, NY 10032, USA
4 Department of Pathology and Neuropathology, Institute of Biomedical Research of Vigo (IBIV), University Hospital of Vigo (CHUVI), Vigo,
36200, Spain
5 Department of Neurology, Hospital Universitari i Politècnic La Fe, València, 46026, Spain, and Biomedical Network Research Centre on
Neurodegenerative Disorders (CIBERNED), Instituto de Salud Carlos III, Madrid, 28029, Spain
6 Neuromuscular Disorders Clinic, Department of Neurology, Hospital Universitari Vall d’Hebron, Institut de Recerca, Universitat Autònoma de
Barcelona, Barcelona, 08035, Spain
7 Department of Pathology and Cell Biology, Columbia University Medical Centre, New York, NY 10032, USA
8 Centro Nacional de Análisis Genómico, Barcelona, 08028, Spain
9 Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague, Prague, 12808, Czech Republic
* These authors contributed equally to this work.
Deceased.
#
These authors contributed equally to this work.
†
Correspondence to: Ramon Martı́, PhD,
Research Group on Neuromuscular and Mitochondrial Disorders,
Vall d’Hebron Institut de Recerca, VHIR, Universitat Autònoma de Barcelona,
Passeig Vall d’Hebron, 119-129
08035 Barcelona, Spain
E-mail: [email protected]
Correspondence may also be addressed to: Michio Hirano, MD, Department of Neurology, Columbia University Medical Centre, 630 West 168th
Street, P&S 4-423, New York, NY 10032, USA. E-mail: [email protected]
Downloaded from http://brain.oxfordjournals.org/ at Universidad de Valencia on May 13, 2013
Maria J. Melià,1,2,* Akatsuki Kubota,3,* Saida Ortolano,4,* Juan J. Vı́lchez,5 Josep Gámez,6
Kurenai Tanji,7 Eduardo Bonilla,3,7,† Lluı́s Palenzuela,1,2 Israel Fernández-Cadenas,1
Anna Přistoupilová,8,9 Elena Garcı́a-Arumı́,1,2 Antoni L. Andreu,1,2 Carmen Navarro,2,4
Michio Hirano3,# and Ramon Martı́1,2,#
In 2001, we reported linkage of an autosomal dominant form of limb-girdle muscular dystrophy, limb-girdle muscular dystrophy
1F, to chromosome 7q32.1-32.2, but the identity of the mutant gene was elusive. Here, using a whole genome sequencing
strategy, we identified the causative mutation of limb-girdle muscular dystrophy 1F, a heterozygous single nucleotide deletion
(c.2771del) in the termination codon of transportin 3 (TNPO3). This gene is situated within the chromosomal region linked to
the disease and encodes a nuclear membrane protein belonging to the importin beta family. TNPO3 transports serine/argininerich proteins into the nucleus, and has been identified as a key factor in the HIV-import process into the nucleus. The mutation
is predicted to generate a 15-amino acid extension of the C-terminus of the protein, segregates with the clinical phenotype, and
is absent in genomic sequence databases and a set of 4200 control alleles. In skeletal muscle of affected individuals, expression of the mutant messenger RNA and histological abnormalities of nuclei and TNPO3 indicate altered TNPO3 function. Our
Received November 5, 2012. Revised January 21, 2013. Accepted February 7, 2013. Advance Access publication March 29, 2013
The Author (2013). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For Permissions, please email: [email protected]
55
March 29th, 2013
LGMD1F is caused by a TNPO3 mutation
Brain 2013: 136; 1508–1517
| 1509
results demonstrate that the TNPO3 mutation is the cause of limb-girdle muscular dystrophy 1F, expand our knowledge of the
molecular basis of muscular dystrophies and bolster the importance of defects of nuclear envelope proteins as causes of
inherited myopathies.
Keywords: limb-girdle muscular dystrophy 1F; LGMD1F; TNPO3; transportin 3; c.2771del mutation
Abbreviation: LGMD = limb-girdle muscular dystrophy
Introduction
Materials and methods
Patients
The reported genealogical investigation of the LGMD1F family (Gamez
et al., 2001) disclosed a common ancestor born in south-eastern Spain
two generations before the oldest living members. The largest branch
originated from Subject II-3 (Gamez et al., 2001) and includes 32
patients with LGMD1F, of whom 28 have been closely followed in
our centre (University Hospital La Fe, València, Spain). Functional activity was assessed using the Brooke score (from 1: normal; to 6: no
function for upper extremity) (Brooke et al., 1981) and the Vignos
score (1: able to climb stairs without help; to 10: bedridden for
lower limb function) (Vignos et al., 1963). Muscle strength was
graded using the Modified Medical Research Council (MMRC) scale.
Whole-body muscle imaging was performed on a 1.5 T or 3 T MRI
scanner. Abnormal muscle signal intensity was ranked according to
Mercuri scale (Mercuri et al., 2002): 1, normal appearance; 2, motheaten appearance with scattered small areas of increased signal involving 530% of muscle volume; 3, moderate involvement (a late motheaten appearance with numerous discrete areas of increased signal
with incipient confluence, involving 30–60% of muscle volume);
4, severe involvement (washed-out or fuzzy appearance due to confluent areas of increased signal, or complete muscle replacement by
connective tissue and fat with only a rim of fascia and neurovascular
structures).
All pedigree identifiers in this report refer to the updated family tree
shown in Supplementary Fig. 1, unless otherwise indicated.
The limb-girdle muscular dystrophies (LGMDs) comprise a group
of genetically heterogeneous disorders characterized by progressive and predominantly proximal muscle weakness with histological signs of degeneration and regeneration in muscle (Bushby,
2009). As a result of molecular characterization and improved
clinical criteria, the classification and nomenclature of LGMDs
have evolved over the last two decades. The canonical categorization of LGMD into autosomal dominant-LGMD (LGMD1) and
autosomal recessive (LGMD2) forms is being refined by a classification based on the affected proteins and their correspondent
genes (Nigro et al., 2011)
In 2001, we reported the clinical and morphological phenotype
of a novel form of autosomal dominant-LGMD affecting 32 individuals in a large Spanish kindred spanning five generations
(Gamez et al., 2001). Clinically, the disorder was characterized
by muscle weakness primarily affecting the pelvic and shoulder
girdles with a wide variability in the age at onset (1–58 years
old), disease progression rate and severity. The disease generally
ran a benign clinical course, but some individuals with childhood
or juvenile onset manifested severe widespread myopathy leading
to wheelchair dependency and respiratory insufficiency. Additional
clinical features of LGMD1F as well as more detailed descriptions
of its time-course and pattern of muscle involvement are presented here.
Initially, the presence of rimmed vacuoles and filamentous
inclusions in myofibres of affected subjects prompted us to consider a diagnosis of hereditary inclusion body myopathy (Huizing
and Krasnewich, 2009). Hereditary inclusion body myopathy typically presents as an autosomal recessive trait and is due to mutations in GNE (9p13.3; Genebank NM_005476); however, a few
cases of autosomal dominant hereditary inclusion body myopathy
have also been described and linked to chromosome 17p13.1
(Martinsson et al., 1999) (IBM3 OMIM #605637) or to 7q22.131.1 (Lu et al., 2012) (IBM4). Hereditary inclusion body myopathy
was ruled out in our family because initial genetic analysis using
simple sequence repeat markers indicated that the disorder was
not linked to hereditary inclusion body myopathy. Our subsequent
studies using genome-wide markers demonstrated a novel locus
for this autosomal dominant-LGMD at the chromosomal locus
7q32.1-32.2, between markers D7S1822 and D7S2519, containing 66 genes. These data confirmed that this family has a genetically distinct form of autosomal dominant-LGMD that was
classified as LGMD1F (Palenzuela et al., 2003) (OMIM
#608423). However, the identity of the mutant gene has been
elusive so far, despite attempts to find it following different
strategies. Here, using a whole genome sequencing approach,
we have identified the causative mutation of the LGMD1F, a
single nucleotide deletion in the termination codon of transportin
3 (TNPO3). The histochemical and ultrastructural findings, together with the molecular results at DNA, RNA and protein
levels, fully support the pathogenic role of this mutation in
LGMD1F.
Muscle biopsies
Muscle biopsies from the deltoid or vastus lateralis from 5 of the 32
affected individuals of the family had been performed in the years
1993 and 1994 under informed consent (Gamez et al., 2001) and
stored at 80 C at the Neurological Tissue Biobank of Vigo
University Hospital. Frozen muscle specimens from Subjects IV-6, IV11 and IV-21 (Supplementary Fig. 1) (Gamez et al., 2001) were
retrieved from the Biobank and further studied by light microscopy.
For ultrastructural studies, original electron microscopy micrographs
were re-examined. Stored Epon-embedded blocks were used to
obtain new ultrathin sections, and were studied under a Philips
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| Brain 2013: 136; 1508–1517
CM100 transmission electron microscope equipped with a digital
camera and the ITEM SYSTEM VELETA (FEI Company) software.
DNA and RNA isolation and
complementary DNA synthesis
DNA was extracted from anticoagulated blood from affected and
unaffected individuals using a standard phenol–chloroform method.
RNA was extracted from skeletal muscle biopsies from Subjects IV-6
and IV-21 and one unrelated healthy control using the RNeasy Kit
(QIAGEN), and treated with the deoxyribonuclease I, amplification
grade (Invitrogen) to eliminate any traces of DNA. Then, complementary DNA was synthesized using the High Capacity complementary
DNA Reverse Transcription kit, which uses random hexamers
(Applied Biosystems).
Sequencing libraries were constructed according to the TruSeqTM DNA
sample preparation protocol (Illumina) with minor modifications, in
particular the double size selection. Two micrograms of genomic
DNA were fragmented with a Covaris E210 and size selected to
300–700 bp. Resulting fragments were end-repaired, adenylated,
ligated to Illumina paired-end adaptors and size selected to very
tight sizes using an E-Gel (Life Technologies). Size selected adapter-insert fragments (two insert sizes: 430 bp and 460 bp) were amplified with 10 PCR cycles and sequenced on an Illumina HiSeq 2000
platform with paired end run of 2 100 bp. Base calling and quality
control was performed on the Illumina RTA sequence analysis pipeline.
Sequence reads were trimmed to the first base with a quality over 30
and mapped to Human genome build hg19 (GRCh37) using GEM
mapper (Marco-Sola et al., 2012), allowing up to four mismatches.
Reads not mapped by GEM mapper (4%) were submitted to a last
round of mapping with BFAST (Homer et al., 2009). Results were
merged and only uniquely mapping non-duplicate read pairs were
used for further analyses. SAM tools suite version 0.1.18 (Li et al.,
2009) with default settings was used to call single nucleotide variants
and short indels. Variants on regions with low mappability (Derrien
et al., 2012), with read depth 510 or with strand bias
P-value 5 0.001 were filtered out. The population frequency of the
variants was assessed by comparing to several databases: the 1000
Genomes Project (http://www.1000genomes.org/), NHLBI Exome
Sequencing Project (ESP) release ESP5400 (http://evs.gs.washington.
edu/EVS/), and our internal database of sequence variants identified in
a set of 4100 control samples). The effect prediction was performed
with Annovar version 2011 Dec20 (Wang et al., 2010) and snpEff
version 2.0.5d (Cingolani et al., 2012).
Dideoxy-DNA sequencing
DNA extracted from blood was used to confirm the segregation of the
genotype and the phenotype. A 856 bp fragment encompassing the
last coding sequence of the TNPO3 gene was PCR-amplified (forward,
5’-TCCTCAGTCAAGGACCAACCTACCT-3’; reverse, 5’-TCCTGTAAG
GGCCAAGCATCCCT-3’), and the product was purified (ExoSAP-IT ,
Affimetrix) and sequenced using the dideoxy method (BigDye
Terminator v3.1 Cycle Sequencing kit, Applied Biosystems). In
order to analyse the sequences of RNA species, complementary
DNA obtained from skeletal muscle of affected and unaffected individuals was PCR-amplified (644 bp fragment, exons 20–24,
primers forward 5’-TCTACTACCCTGGACCACCG-3’ and reverse
5’-GCGCTGATTTTCCCTCACAC-3’) and the resulting fragments
were sequenced.
Polymerase chain reaction–restriction
fragment length polymorphism analysis
A 629 bp fragment was PCR-amplified (Forward primer
5’-TCTACTACCCTGGACCACCG-3’
and
Reverse
primer
5’-CACACCCCCAAACAGGAACT-3’) from skeletal muscle complementary DNA from Subjects IV-6, IV-21 and one unrelated healthy
control subject. The products were digested with the restriction
enzyme SfaNI (New England Biolabs). The wild-type sequence of the
629 bp amplicon contains a single SfaNI target generating two fragments: 617 bp + 12 bp. The c.2771del mutation generates an additional target, producing the expected restriction fragment pattern of
400 bp + 216 bp + 12 bp. The fragments were resolved by electrophoresis in a 2% agarose gel, visualized by ethidium bromide staining,
and the bands were quantitated by densitometry using ImageJ
software.
Western blot
Frozen muscle biopsy samples were homogenized in lysis buffer
containing 0.25% NP-40 with protease inhibitor cocktail (cOmplete
Mini , Roche Diagnostic), and after centrifugation, supernatants
were collected. Concentrations of protein in the supernatants were
measured by bicinchoninic acid assay. Aliquots containing 40 mg protein were separated by SDS-PAGE and transferred to a membrane.
After blocking with PBS containing 0.5% skimmed milk, the membrane was incubated at 4 C overnight with primary antibodies: antiTNPO3 antibody (ab54353, Abcam 1:50) and anti-beta-actin antibody
(20536-1-AP, Proteintech, 1:1000). The immunoprobed membrane
was washed with PBS containing 0.5% Tween 20 three times, and
was incubated for 1 h at room temperature with peroxidase-conjugated anti-mouse IgG antibody or anti-rabbit IgG antibody. After
incubation with secondary antibodies, the membrane was washed
with PBS containing 0.5% Tween 20 three times, and was developed
with ECL Prime Western Blotting Detection Reagents (GE
Healthcare). The membrane was imaged with G:BOX Chemi IR6
(SYNGENE).
Whole genome sequencing
M. J. Melià et al.
Anti-TNPO3 and 4’,6-diamidino-2phenylindole staining
Six-micrometre thick sections of frozen muscle were fixed in ice-cold
acetone for 10 min, incubated for 1 h with 1% bovine
serum albumin, and stained at 4 C for overnight with murine antiTNPO3 antibody (ab54353, Abcam) at a concentration of 5 mg/ml.
Specimens were then incubated for 1 h with sheep biotinylated antimouse IgG antibody (RPN1001, GE Healthcare, 1:100) followed by 1 h
with streptavidin-fluorescein (RPN1232, GE Healthcare, 1:250),
mounted, and stained with 4’,6’-diamidino-2-phenylindole dihydrochloride (DAPI) using VECTASHIELD Mounting Medium with
DAPI (Vector Laboratories). The stained sections were examined
with a confocal microscope (Leica TCS SP5 II , Leica
microsystems), and images were obtained with LAS AF (Leica
microsystems).
57
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Brain 2013: 136; 1508–1517
Results
Clinical assessments
stage 8 at the fifth and seventh decade. Three additional patients
reached Vigno stages 6 and 7 in their forties through sixties. Two
patients died suddenly at age 57 and 78 due to causes unrelated
to the myopathy.
Laboratory investigations provided similar results to those previously reported (Gamez et al., 2001); however, electromyography
in the current series frequently showed spontaneous activity, and,
in 3 out of 8 patients, displayed clear motor neurogenic features.
In two patients who presented with intense fatigue, ptosis and
transient dysphagia, repetitive stimulation tests and single-fibre
electromyography disclosed no abnormalities in neuromuscular
transmission. Serum creatine kinase levels were also consistent
with the previous report: 40% of the cases showed elevated creatine kinase levels (4500 U/l, maximum 2200). No correlation
between clinical severity and creatine kinase levels was identified.
Muscle MRI demonstrated variable involvement of scapular and
pelvic-femoral muscles, as well as lower leg muscles
(Supplementary Fig. 3). A characteristic relationship between
muscle MRI abnormalities and degree of impairment was observed
with intensity of the MRI signal changes correlating well with the
severity of the clinical involvement. In general, scapular-humeral
girdle muscles were much better preserved than pelvic-femoral
and leg muscles. The percentage of cases with moderate (3) or
severe (4) Mercuri scores by muscle group are: (i) scapular girdle:
teres major (80%), pectoral (64%), infraspinatus and serratus anterior (55%), deltoids (46%); (ii) lumbar: paraspinal (90%), abdominal oblique (55%) and rectus abdominus (55%); (iii) thigh:
sartorius (100%), vastus lateralis, intermedius and medialis (73%),
biceps femoris and semitendinosus (55%); and (iv) lower leg:
peroneal (91%), gastrocnemius (91%), soleus (73%) and tibialis
anterior (70%). Correlations between the clinical severity and the
degree of MRI muscle affectation are presented in Supplementary
Fig. 3. Subject V-9 represents a mildly symptomatic subject without overt clinical weakness but with Mercury stage 3 abnormalities
in lumbar paraspinal, sartorius and peronei muscles. Subject V-7
represents a moderately affected subject (Vignos scale 5) showing
a widespread muscle involvement (Mercuri stage 3) of paravertebral and abdominal lumbar muscles, anterior and posterior thighs
and diffuse lower leg muscles. Finally, Subject IV-26 corresponds
to a severely affected patient (Vignos rating of 7) manifesting
Mercuri 3 and 4 grade abnormalties in scapular, lumbar, thigh
and lower leg muscles.
The cohort reported here comprises 30 individuals spanning
Generations III to VI; 13 were included in a previous study
(Gamez et al., 2001). They presented with limb-girdle and distal
muscle weakness with variable distribution, severity, and rate of
progression (Supplementary Table 1). Based on age-at-onset, a
predominant group of juvenile-onset (onset before age 15) was
delineated from an adult-onset form starting in the third and
fourth decades (Gamez et al., 2001). Although a similar distribution was observed in the present cohort, the difference between
juvenile- and adult-onset groups was not pronounced.
Six patients in the fifth and sixth generations had infantile onset
disease characterized by a mild delay in motor skill (independent
walking was never delayed beyond age 22 months), followed by
difficulty rising from the floor to a standing position and climbing
stairs without the aid of hand rails. Running and jumping were
difficult or impossible. Some manifested an abnormal gait with a
mixture of waddling and distal leg weakness. When walking or
attempting to stand on heels, patients also demonstrated a peculiar posture of the feet with elevation of the big toe, foot drop and
weight-bearing on the lateral soles (Supplementary Fig. 2). In addition, all patients had thin legs and thenar muscle atrophy. Two
patients presented early contractures at the heels, knees, and
elbows with rigid spine and scoliosis reminiscent of EmeryDreifuss muscular dystrophy, but lacking cardiac involvement.
Common initial symptoms in the late-childhood and adolescent
group were difficulty running and playing sports. Overt symptoms
of pelvic-femoral weakness, such as difficulty in rising from the
floor or climbing steps, were also frequent. One patient had exercise intolerance, myalgia and fatigue reminiscent of a metabolic
myopathy.
Symptoms of pelvic-girdle weakness were the most common
presentation in late-onset patients. Weakness and atrophy of
shoulder girdle muscles appeared in only 70% of cases, always
in later or advanced stages of the disease, and usually showing
less degree of involvement than muscles of pelvic-femoral and
axial territories. While both mild (Brook scale 1–2) and severe
(Brook scale 3–4) cases showed minor scapular winging
(Supplementary Fig. 2), prominent scapula alata was never
observed.
Distal muscle involvement was more frequent (at least 24 of 30
patients) than previously reported. In hands, thenar muscle atrophy was observed in all 24 carefully assessed patients and a high
proportion of cases reported difficulty grasping a pencil or opening
jars. In lower leg, subclinical muscle weakness was often revealed
by asking patients to stand on their heels. Other symptoms associated with the disease were: mild ptosis (five cases), transient
dysphagia (nine cases) and episodic vertigo and ataxia (eight
cases). Three cases had respiratory muscle involvement; all
required non-invasive nocturnal ventilatory support.
The course of the disease was highly variable. The two most
severe cases with an Emery-Dreifuss-like phenotype were wheelchair-bound in the third decade. Two other cases reached Vigno
| 1511
Histological studies
Previous analyses of muscle biopsies from five people affected
with LGMD1F had revealed increased variability of fibre size and
shape, increased endo- and peri-mysial connective tissue, scattered
degenerating fibres, occasional central nuclei, abnormal intermyofibrillar network with abnormal Z bands, rimmed vacuoles and
abnormally increased mitochondria with rare paracrystalline inclusions (Gamez et al., 2001). These histological features are similar
to those recently reported in the same family (Cenacchi et al.,
2012). New analyses of muscle biopsies from seven affected patients (Supplementary Fig. 1) confirmed the described abnormalities in myofibres and connective tissue. In addition, we observed
unusually enlarged nuclei with central pallor (Fig. 1). These nuclear
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Subject IV-6. In A, abnormal myonuclei with an ‘empty’ appearance (arrows) (630). Scale bar = 20 mm. In B, note three abnormal nuclei
(arrows) within a myofibre at higher magnification (1000). Scale bar = 10 mm. (C and D) Electron micrographs showing non-branching
tubular filaments 18–20 nm in diameter within a muscle fibre (Subject IV-6). Note myelin and membranous bodies surrounding filaments
(C), which are characteristic of rimmed vacuoles. Original magnifications: 21 000 and 35 000. Scale bars = 0.5 mm.
abnormalities were identified in all seven biopsies in 11.0–25.8%
of muscle fibres (Subject III-14: 18.8%; Subject IV-6: 16.7%;
Subject IV-11: 14.8%; Subject IV-18: 16.0%; Subject IV-21:
17.4%; Subject IV-36: 25.8%; Subject V-14: 11.0%). These percentages were derived from counts of the number of affected
nuclei and the total number of fibres within each biopsy in haematoxylin–eosin stained slides, using a histometric program (Leica
Application Suite v 3.8.0). We have not observed these nuclear
abnormalities in other myopathies including Duchenne muscular
dystrophy, sarcoglycanopathies, Emery-Dreifuss muscular dystrophy due to emerin or lamin A/C mutations, or FHL1 dystrophy.
Ultrastructurally, filamentous inclusions, 18 to 20 nm in diameter,
were detected within nuclei or in the cytosol of a minority of fibres
in two out of five biopsies (Subjects IV-6 and IV-21, Fig. 1). In
three biopsies, corresponding to Subjects IV-6, IV-18 and IV-21,
light microscopy showed rimmed vacuoles and electron
microscopy revealed autophagic vacuoles with prominent pseudomyelin structures, membranous whorls and dense bodies.
Immunocytochemical stains for desmin, dystrophin, sarcoglycans,
tau, ubiquitin, and amyloid-b proteins, did not show significant
alteration or accumulation in muscle fibres.
Genetic and molecular studies
Initial strategies to identify the genetic cause of the disease
included sequencing of candidate genes. Among them, FLNC,
encoding filamin c, was extensively investigated because
mutations in this gene cause autosomal dominant myofibrillar myopathy (Vorgerd et al., 2005) (OMIM #609524). Studies included
dideoxy-DNA sequencing of FLNC exons, flanking introns and
promoter regions, Southern and northern blot analyses, and
immunohistochemical staining and western blot analyses of
muscle biopsies with anti-FLNC antibodies, and revealed normal
results compared to controls (data not shown) thereby excluding
FLNC as the causative gene.
Dideoxy sequencing of 65 additional genes within the region
failed to reveal potentially pathogenic mutations, although the
presence of heterozygous changes was difficult to be completely
ruled out in some of the electropherograms due to suboptimal
quality. Comparative Genomic Hybridization (CGH, NimbleGen
Systems Inc.) across the linked region excluded genome copy
number variations. Segmentation values across the chromosome
7 regions of interest and other chromosomes showed no differences in DNA from two control subjects and two affected individuals, thereby excluding DNA copy number alterations as the cause
of the disease (data not shown).
Because studies at the DNA level were unrevealing, we performed analyses of messenger RNA levels. Expression of 36
genes included in the critical region was analysed in RNA extracts
from skeletal muscle of affected members of the family and unaffected unrelated subjects, using a TaqMan Custom Array 384well microfluidic card (Supplementary Fig. 4 and Supplementary
Table 2). No significant differences could be detected in the expression of the genes analysed, except for a moderate increase of
Figure 1 Light and electron microscopy findings in muscle biopsies. (A and B) Haematoxylin and eosin staining muscle from affected
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DNA revealed the coexistence of both mutated and wild-type
transcripts in similar amounts, according to the sizes of the
peaks of the two overlapped sequences observed in the electropherograms (Fig. 2). This result was confirmed by PCR-restriction
fragment length polymorphism analysis, which indicated that 61–
64% of the TNPO3 messenger RNA of the two affected individuals contained the mutant form (Fig. 2). Retrospective review of
the real-time PCR results obtained in the microfluidic cards
(Supplementary Fig. 4) confirmed that TNPO3 was expressed in
skeletal muscle of affected and non-affected persons at similar
levels. Taken together, these results demonstrate that the mutated
messenger RNA is stable and does not undergo RNA decay.
We performed western blot analysis of biopsied muscle samples
from four affected subjects and two unaffected control subjects, to
assess changes in amount and molecular weight of TNPO3 protein
(Fig. 2). The TNPO3 mutation in the family disrupts the termination codon, and is predicted to extend the C-terminus of TNPO3
by 15 amino acids. Using an anti-TNPO3 antibody that recognizes
an N-terminus epitope, present in both normal and mutant
TNPO3, western blot analysis showed a single band at the same
level in muscle from control subjects and affected individuals, and
no extra bands were observed in muscles from affected subjects.
However, the 15 amino acid size difference between normal and
mutant TNPO3 is likely insufficient to distinguish the two proteins
by western blot. Relative to control subject muscles, the amount
of muscle TNPO3 normalized to beta-actin was increased in one
affected subject (Subject IV-36), and decreased in the other three
(Subjects V-14, III-14 and IV-18); therefore, there were no significant difference in TNPO3 quantity in mutant versus normal tissue.
To assess the effects of the TNPO3 mutation on TNPO3 cellular
localization, we performed immunohistochemistry of muscle tissue
with anti-TNPO3 antibody. Control muscle stained with antiTNPO3 antibody showed clear nuclear staining and that colocalized with DAPI (Fig. 3I). In muscle of affected individuals, TNPO3
immunostaining was also observed within nuclei, but was unevenly distributed and often limited to the periphery of nuclei,
(Fig. 3C and F).
Discussion
NRF1 transcripts and a pronounced increase of LEP transcripts, in
skeletal muscles from affected persons. Because LEP, encoding
leptin, is highly expressed in adipose tissue (Meier and Gressner,
2004), we suspected that elevated LEP expression reflected fat
replacement of affected muscle, rather than a primary pathogenic
alteration. We analysed the expression levels of ADIPOQ, which is
expressed exclusively in adipose tissue (Maeda et al., 1996), and
also found increased levels of this marker of fat tissue in muscles
from affected subjects (Relative quantification (RQ) median; interval: 73.8; 23.9–339.0; n = 5) as compared with levels in unaffected subjects (RQ median; interval: 3.0; undetectable -10.3;
n = 6). The levels of ADIPOQ transcripts closely correlated with
values for LEP messenger RNA (P 4 0.001; Spearman correlation
coefficient R = 0.991), thus supporting the notion that increases in
LEP transcript reflected fatty replacement of muscle.
After our initial efforts to find the genetic cause of LGMD1F
failed, we applied a more powerful strategy, whole genome
sequencing analysis of DNA from one affected individual
(Subject III-12, Supplementary Fig. 1). After intersecting the results
of whole genome sequencing with the results from previous linkage analysis (Palenzuela et al., 2003) (chromosome 7: 126 287
120–129 963 917), 3888 variants (3125 single nucleotide variants
and 763 indels) were identified, from which 718 were novel, not
present in the dbSNP database, build 135 (http://www.ncbi.nlm.
nih.gov/projects/SNP/). Additional criteria based on the dominant
inheritance of the disease, the population frequency of the variants and effect prediction (see ‘Materials and methods’ section),
allowed us to rule out all but one of these variants, a heterozygous
mutation in the termination codon of the TNPO3 gene, encoding
transportin 3, a protein involved in the translocation of proteins
from the cytoplasm to the nucleus (Brass et al., 2008; Cribier
et al., 2011). The mutation (c.2771del, reference sequence
GeneBank NM_012470.3, Fig. 2) is a single adenine nucleotide
deletion in the TAG stop codon, common to the two protein isoforms encoded by the gene. The del-A results in conversion of
TAG to TGC codon, encoding cysteine, and extension of the reading frame by 15 codons to a downstream of the termination-signal
within the transcript. Thus, the frameshift leads to the predicted
mutated TNPO3 protein with 15 additional amino acids at the
C-terminus [p.(*924Cysext*15) for isoform 1]. The retrospective
analysis of the sequences of this gene revealed that this heterozygous mutation had been missed in the past due to the poor
quality of the electropherograms. Then, we performed new
dideoxy sequence analysis of TNPO3, which demonstrated presence of c.2771del in each of the 29 clinically affected individuals
and absence of the mutation in all 20 clinically unaffected relatives
tested. Thus, the sequence data indicate that the mutation segregates with the linked chromosome 7q32.1-32.2 region (Palenzuela
et al., 2003) and with the phenotype (Supplementary Fig. 1).
To investigate whether the mutated messenger RNA was
expressed in skeletal muscle of the affected individuals, complementary DNA was generated using RNA from two skeletal muscle
samples (Subjects IV-6 and IV-21, Supplementary Fig. 1), and
the 3’-end fragment containing the native stop codon (common
to the 3 transcripts described for the gene, TNPO3 gene entry in
the NCBI, http://www.ncbi.nlm.nih.gov/gene/23534) was PCRamplified. Sequence analysis of this amplified complementary
| 1513
LGMD1F is one of the nine autosomal dominant forms of LGMD.
Causative genes had been identified for only five forms of autosomal dominant-LGMD: MYOT (LGMD1A, OMIM #159000),
LMNA (LGMD1B, OMIM #159001), CAV3 (LGMD1C, OMIM
#607801), DES (LGMD1D, OMIM *125660), and DNAJB6
(LGMD1E, OMIM #603511) (Bushby, 2009; Sarparanta et al.,
2012) (see GeneReviewsTM LGMD Overview). In general, these
disorders are characterized by adult-onset and milder clinical
phenotypes than LGMD2. Although, most individuals harbouring
mutations in these genes fulfil the diagnostic criteria for LGMD,
some manifest a wider spectrum of clinical phenotypes. The extreme example is LMNA mutations, which have been associated
with a broad spectrum of clinical conditions including Dunnigan
lipodystrophy, autosomal dominant Emery-Dreifuss muscular dystrophy, cardiomyopathy, Charcot–Marie–Tooth disease and
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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 + 216 bp + 12 bp. Densitometric analysis of the bands showed
that the mutated messenger RNA was 64% (Subject IV-6) and 61% (Subject IV-21) of total TNPO3 messenger RNA. (D) Western blot of
muscle specimens from affected subjects and controls. Muscle specimens from four subjects with LGMD1F and two control subjects were
analysed by western blot. The anti-TNPO3 antibody showed a clear band at approximately 100 kDa in each lane. No differences in the
position of bands and no extra bands were observed. There were no significant differences in the amounts of TNPO3 normalized to
beta-actin between affected subjects and controls.
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| 1515
affected individuals (Subject IV-36: A–C, Subject V-14: D–F) and two control subjects (one not shown) (G–I) were observed under a
confocal microscope. Each specimen was stained both with anti-TNPO3 antibody (A, D and G) and by DAPI (B, E and H), and merged
images were generated (C, F and I). TNPO3 staining colocalized with DAPI in control subjects (I). In affected individuals, signals of TNPO3
were also observed within nuclei, but were unevenly distributed (C and F). Scale bar = 40 mm.
Hutchinson-Gilford progeria (Worman et al., 2009; Bertrand et al.,
2011).
Several lines of evidence strongly support pathogenicity of the
TNPO3 mutation in this family with autosomal dominant-LGMD:
(i) TNPO3 resides within the chromosome 7q32.1-32.2 locus for
LGMD1F; (ii) the mutation segregates with the phenotype; (iii) the
microdeletion is absent in publicly available genomic sequence
databases (dbSNP build 135, 1000 Genomes Project and 5400
NHLBI exomes) and in our set of 4200 Spanish control alleles
indicating that all control individuals harbour the canonical
TNPO3 TAG termination codon in homozygosity at the position
128 597 311 of the chromosome 7; (iv) the mutation in the termination codon of TNPO3 is predicted to extend the coding
sequence at the 3’-end of the messenger RNA and to generate
an aberrant protein; (v) the mutated messenger RNA is expressed
in the muscle of the affected individuals; and (vi) the detection of
histologically abnormal muscle nuclei with atypical nuclear
filaments, anomalous TNPO3 immunoreactivity and irregular
membranes. These morphological changes of myocyte nuclei
indicate that the TNPO3 c.2771del mutation alters nuclear functions, which is consistent with the putative role of the TNPO3 in
transport of proteins across the nuclear membrane. We observed
similar levels of TNPO3 transcript in skeletal muscle of three
healthy control subjects and in five affected subjects
(Supplementary Fig. 4). It is likely that the mutant protein, which
is predicted to contain 15 additional amino acids at the C-terminus,
is expressed in skeletal muscle and exerts a dominant toxic effect.
Although there is evidence that TNPO3 is expressed in skeletal
muscle (BioGPS portal for annotation resources (http://biogps.org)
(Su et al., 2004), the role of TNPO3 in muscle is currently unknown. TNPO3 was originally identified as TNP-SR2, which encodes a nuclear membrane protein belonging to the importin beta
family and transports serine/arginine (SR) rich proteins into the
nucleus (Lai et al., 2000, 2001). TNPO3 was subsequently identified by genome-wide RNA interference knockdown as a HIV-dependency factor required for HIV1 infection at a stage between
reverse transcription and integration of HIV in human cells (Brass
et al., 2008; Konig et al., 2008). The protein mediates nuclear
Figure 3 Anti-TNPO3 and DAPI staining of muscle from affected individuals and controls. Immunofluorescence-stained muscle from two
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cases with a congenital myopathy phenotype and a variable
course, which can evolve into a severe and rapid progressive
phenotype. Interestingly, these severely affected patients also
manifested early joint and axial contractures similar to EmeryDreifuss syndrome, which raises the possibility of pathogenic
mechanisms distinct from those involved in the typical cases. In
addition, we have also observed very benign patients without
complaints of weakness, but rather atypical features such as
myalgia, exercise intolerance, and fatigue, mimicking a metabolic
myopathy. This broad scenario of clinical nuances highlights the
need to deepen the clinical evaluation of this extensive pedigree
and other potential families with similar gene defects.
In summary, in this report, we have provided an extensive
update of the clinical and morphological features of LGMD1F
and have identified a microdeletion mutation in the TNPO3
gene as a cause of this disorder. This finding expands our knowledge on the genetic bases of muscular dystrophies and suggests
that other proteins of the nuclear envelope compartment may play
a primary role in the pathogeneses of muscular dystrophies and
other skeletal muscle-related disorders.
Acknowledgements
The authors thank the members of the family studied in this work
for their collaboration, and Gisela Nogales-Gadea for scientific
assistance.
Funding
This work was supported by the Spanish Instituto de Salud Carlos
III [PS09/01591 to R.M., PI10/02628 to C.N., PI11/0842 to S.O.,
PI10/01970 to J.G., RD09/0076/00011 to the activities of
Neurological Tissue Biobank, BIOBANCO del CHUVI]; the
International Rare Diseases Research Consortium [SpainRDR]; the
Conselleria de Economia e Industria, Xunta de Galicia [contract
Isidro Parga Pondal to S.O.]; the U.S. National Institutes of
Health (NIH) [R01 AR47989 to M.H.]. The CNAG thanks for
core funding from the Spanish Ministerio de Economia y
Competitividad and the Generalitat de Catalunya - Departament
de Salut and Departament d’Economia i Coneixement. M.H. and
A.K. also acknowledge support from NIH grants R01 HD057543
and R01 HD056103 from NICHD and the Office of Dietary
Supplements (ODS), as well as U54 NS078059 from NINDS and
NICHD, and from the Muscular Dystrophy Association USA.
import of the HIV pre-integration complex by binding the viral
integrase, both in dividing and non-dividing cells (Christ et al.,
2008). The C-terminus domain (CTD) of TNPO3 appears to be
required for interactions with HIV1 integrase (Larue et al., 2012);
therefore, the abnormal extension of the CTD domain is likely to
interfere with its transport function.
Because specific combinations of SR proteins are required for
messenger RNA splicing and post-transcriptional processing
(Bjork et al., 2009), the TNPO3 mutation may alter muscle transcripts raising the possibility that LGMD1F is RNA-mediated
myopathy similar to, but mechanistically distinct from, myotonic
dystrophy (Wheeler and Thornton, 2007; Tang et al., 2012). In
addition, because mutations in the nuclear envelope proteins
emerin and lamin A/C, are known to cause Emery-Dreifuss muscular dystrophy, the TNPO3 mutation causing LGMD1F extends
the genetic spectrum of nuclear envelope-related myopathies. In
support of this notion is the observation of filamentous inclusions
and rimmed vacuoles in all three diseases (Fidzianska et al., 2004).
We have noted filaments of 18 to 20 nm diameter both in nuclei
and within the cytosol of myofibres of affected individuals.
Filaments of similar thickness have been observed in several
muscle disorders and are ascribed to accumulations of proteins,
such as beta-amyloid, tau protein and ubiquitin (Askanas and
Engel, 2006; Askanas et al., 2009). Myonuclear breakdown
would entail the fragmentation of the nuclear membrane and contribute to the formation of pseudomyelin figures and membranous
whorls, which correspond to the rimmed vacuoles seen by light
microscopy.
Interestingly, autophagic vacuoles, which we have observed in
our affected subjects’ muscle, have been also noted in LGMD1D,
which is due to mutations in DNAJB6. In that disease, the presence of autophagy is due to abnormal protein accumulation,
which confers a dominant toxic function to the autophagic complex that contains the mutated co-chaperone (Sarparanta et al.,
2012). Accordingly, autophagy may be contributing to LGMD1F.
Clinically, LGMD1F was originally described as slowly progressive proximal symmetric weakness with predominantly lower limb
onset, normal to mildly raised creatine kinase activity and myopathic electromyography features (Gamez et al., 2001). Great
variability in age at onset, distribution of muscle involvement
and severity was observed. Two clinical forms were delineated: a
benign adult-onset form presenting in the third decade or later,
and a juvenile form, beginning before age 15 years and leading to
severe functional disability. Relative to the original report, the present study of a cohort of 30 patients with LGMD1F provides a
longer and more systematic follow-up, as well as new information
about affected individuals from younger generations. While confirming the core phenotype of LGMD1F, which is characterized by
pelvic-femoral weakness and less severe and variable shoulder involvement, the new clinical data also demonstrate a broad clinical
spectrum and novel clinical features of the disease. The disorder
usually begins in childhood or adolescence, and less frequently in
adulthood; and typically runs a benign course compatible with a
normal working life. We noted mild hand and lower leg weakness
producing a stereotyped posture while walking or standing on
heels in virtually all patients. In addition, we observed less common
but well-characterized presentations, including infantile-onset
Supplementary material
Supplementary material is available at Brain online.
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combinations of SR proteins associate with single pre-messenger RNAs
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Brass AL, Dykxhoorn DM, Benita Y, Yan N, Engelman A, Xavier RJ, et al.
Identification of host proteins required for HIV infection through a
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Pellegrino RJ. Clinical trial in Duchenne dystrophy. I. The design of
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Cenacchi G, Peterle E, Fanin M, Papa V, Salaroli R, Angelini C.
Ultrastructural changes in LGMD1F. Neuropathology 2012. Advance
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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 desminassociated cytoskeletal network.
Supported by: Telethon Italy, AFM (Association Francaise contre les Myopathies).
Disclosure: Dr. Angelini has received personal compensation for activities with Genzyme as a
member of the Advisory Board. Dr. Peterle has nothing to disclose. Dr. Fanin has nothing to
disclose. Dr. Cenacchi has nothing to disclose. Dr. Nigro has nothing to disclose.
12 febbraio 2013
65
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December 21st, 2012
Neuropathology 2012; ••, ••–••
doi:10.1111/neup.12003
Original Article
Ultrastructural changes in LGMD1F
Giovanna Cenacchi,1 Enrico Peterle,2 Marina Fanin,2 Valentina Papa,1 Roberta Salaroli1 and
Corrado Angelini2,3
Department of Biomedical and Neuromotor Sciences, “Alma Mater” University of Bologna, Bologna, 2Department of
Neurosciences, University of Padova, Padova and 3IRCCS S.Camillo, Venice, Italy
1
A large Italo-Spanish kindred with autosomal-dominant
inheritance has been reported with proximal limb and axial
muscle weakness. Clinical, histological and genetic features
have been described. A limb girdle muscular dystrophy 1F
(LGMD1F) disease locus at chromosome 7q32.1–32.2 has
been previously identified. We report a muscle pathological
study of two patients (mother and daughter) from this
family. Muscle morphologic findings showed increased
fiber size variability, fiber atrophy, and acid-phosphatasepositive vacuoles. Immunofluorescence against desmin,
myotilin, p62 and LC3 showed accumulation of myofibrils,
ubiquitin binding protein aggregates and autophagosomes.
The ultrastructural study confirmed autophagosomal vacuoles. Many alterations of myofibrillar component were
detected, such as prominent disarray, rod-like structures
with granular aspect, and occasionally, cytoplasmic bodies.
Our ultrastructural data and muscle pathological features
are peculiar to LGMD1F and support the hypothesis that
the genetic defect leads to a myopathy phenotype associated with disarrangement of the cytoskeletal network.
Key words: electron microscopy, histopathology, limb
girdle muscular dystrophy 1F, myofibrillar, myopathy.
INTRODUCTION
The limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of hereditary neuromuscular disorders
with predominant or selective weakness in the proximal
limb and/or distal muscles, having an estimated incidence
of 1:100 000.1–3 The clinical phenotype is characterized by a
great variability, ranging from early onset, with a severe
and rapidly progressive clinical course, to milder forms,
with a later onset and a slower progression. Autosomal-
Correspondence: Giovanna Cenacchi, MD, Department of Biomedical and Neuromotor Sciences, Via Massarenti, 9, 40138 Bologna, Italy.
Received 10 July 2012; revised and accepted 8 November 2012.
dominant (AD) families representing less than 10% of the
whole group of LGMDs,1–3 and to date, eight AD forms of
LGMD, have been described.Among the AD forms, a large
Italo-Spanish kindred with LGMD1F has been described
with proximal limb and axial muscle weakness.4,5 Clinical,
histological and genetic features have been described in
5/32 patients. In this family, the disease locus has been
mapped to chromosome 7q32.1–32.2, but no mutation
was detected in filamin C, a possible candidate gene in
this chromosomal region, which encodes for actin binding
protein highly expressed in muscle.5 The clinical and morphological features of two patients with LGMD1F are here
described since the disease was not yet sufficiently characterized to suggest a specific etiologic category.
We report a muscle pathological study of two patients
(mother and daughter) from this Italo-Spanish family.
An ultrastructural and immunofluorescence approach
has been performed to investigate the pathogenetic
mechanism.
MATERIALS AND METHODS
Muscle biopsy histopathology was investigated in one pair
of affected patients (mother, Case 1; daughter, Case 2).
Childhood onset was observed in Case 2 (6–7 years) with a
faster weakness progression; at 22–23 years the patient
showed difficulty in rising with mild respiratory and swallowing impairment. In comparison, in Case 1 clinical symptoms were relatively mild until the age of 32 years; then the
progression rate was slow. Skeletal muscle biopsies were
performed in vastus lateralis after obtain patient consent.
Muscle specimens were oriented, snap-frozen in liquid
nitrogen-chilled isopentane and the cryostat-cut sections
were stained using a panel of routine histochemical
methods: HE, modified Gomori trichrome, reduced
nicotinamide-adenine-dinucleotide-tetrazolium-reductase
(NADH-TR), combined cytochrome oxidase (COX) and
succinic dehydrogenase (SDH), adenosine triphosphatases
(ATPases) and acid phosphatase. Immunofluorescence
analysis for desmin (MAB1698 Chemicon, Temecula, CA,
© 2012 Japanese Society of Neuropathology
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December 21st, 2012
2
G Cenacchi et al.
US; dilution 1:50) and myotilin (RSO34, Novocastra, Newcastle, UK; dilution 1:50) and double-labeling for LC3
(2775,Cell SignalingTechnology,Danvers,MA,US;dilution
1:100) and p62 (Gp62C, Progen Biotechnik, Heidelberg,
Germany; dilution 1:200) were performed using immunofluorescence microscopy. Fresh tissues from each biopsy
were fixed in 2.5% glutaraldehyde in cacodylate buffer,
post-fixed in 1% OsO4 in the same buffer, dehydrated in
graded ethanol, and embedded in araldite. Semithin sections were stained with toluidine blue.Thin sections, stained
with uranyl acetate and lead citrate, were examined with a
Philips 400T transmission electron microscope.
A
B
C
D
RESULTS
Morphologic findings by HE showed increased fiber size
variability; the fibre atrophy was more prominent in Case
2, whereas endo- and perimisial connective tissue, and acidphosphatase-positive areas were more pronounced in Case
1 (Fig. 1). Immunofluorescence for desmin and myotilin
showed accumulation of reaction in myofibrillar structures
in some fibers of both patients (Fig. 2). Double-labelling
for p62 and LC3 demonstrated increased protein aggregates (p62-positive) within some atrophic fibers (Fig. 3);
LC3 labelling, used as a marker of autophagosomes, was
Fig. 1 Muscle biopsy sections stained for
HE (A,B), and acid phosphatase (C,D) of
Case 2 (A,C) and Case 1 (B,D). Note fiber
size variability, diffuse fiber atrophy in
Case 2 (A, B) and accumulations of acid
phosphatase-positive material (C,D). Microscope magnification ¥200.
Fig. 2 Muscle biopsy sections from Case 2
(A,C,D) and Case 1 (B) immunostained for
desmin (A) or myotilin (B–D). Note accumulation of cytoskeletal (desmin) or sarcomeric
(myotilin) proteins, occupying a relatively
large cytoplasmic area of isolated myofibers.
Microscope magnification ¥200.
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December 21st, 2012
3
Ultrastructure of LGMD1F
Fig. 3 Double immunofluorescence analysis on muscle biopsy sections using antibodies against p62 (green) and LC3 (red)
(counterstain of nuclei with 4′,6-diamino-2phenylindole, blue) in Case 2 (A,B) and Case
1 (C,D). Note accumulation of p62-positive
protein aggregates in some atrophic muscle
fibers in both patients.
Fig. 4 Ultrastructural analysis showing
atrophic fiber characterized by many polymorphic mitochondria with paracrystallinelike inclusions (arrow) (A) and myelinoid
body adjacent to the subsarcolemmal
nucleus (B) in Case 2. Myofibrillar alterations lead to architectural disarray of muscle
fibers in Case 1 (C), and to accumulation of
electrondense material of possible Z-linederivation in the subsarcolemmal areas in
Case 2 (D). At higher magnification, the
electron-dense material appears as a
granulo-filamentous pattern (d-inset). Scale
bar = (A) 2000 nm; (B) 1000 nm; (C)
2000 nm; (D) 5000 nm (inset: 2000 nm).
A
B
C
D
mild and diffuse (Fig. 3). The ultrastructural study confirmed fiber atrophy (Fig. 4), abnormal mitochondria accumulations with only rare paracrystalline-like inclusions
(Fig. 4) and autophagosomal vacuoles containing cytoplasmic debris and myelinoid bodies (Fig. 4). No tubulofilamentous cytoplasmic or nuclear inclusions were
detected. Many alterations of myofibrillar component were
easily detected, such as prominent disarray (Fig. 4), rodlike structures with granular aspect (Fig. 4), and occasional
filamentous cytoplasmic bodies. No ultrastructural differences were appreciated between the two cases.
DISCUSSION
The histopathological data and the electron microscopic
findings from our patients extend previous results described
for this LGMD1F family.4,5 In the original report, the morphologic findings were abnormal fiber size with degenerative aspect and prominent rimmed vacuoles.4 COX–SDH
stain showed about 5–30% of COX-negative fibres. The
ultrastructural description has been performed in three
cases.4 That study focused on the presence of both
autophagosomes with cytoplasmic bundles of filaments in
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December 21st, 2012
4
G Cenacchi et al.
one case, and a large amount of degenerating mitochondria,
which were considered a secondary unspecific feature.4
Changes related to mitochondria and autophagosomal
vacuoles are commonly seen in a wide variety of myopathies
such as inclusion body myositis (IBM)6,7 and oculopharyngeal nuscular dystrophy (OPMD).8–10 Whereas abnormalities of p62 and ubiquitin-binding proteins might be signals
of protein degradation, in our cases protein aggregates were
associated with p62 and diffuse LC3. Abnormalities of
NBR1 (neighbor of BRCA1 gene 1), a novel autophagyassociated protein, could be a useful tool to further study
chronic progressive myopathies.7 We did not identify in our
patients’ biopsies, neither nuclear nor cytoplasmic tubulofilament inclusions, which are considered specific for IBM if
associated with the presence of rimmed vacuoles.6,7 We did
not see intranuclear tubulofilaments arranged in tangles or
palisades which are described in the OPMD.9,10 The present
ultrastructural observations highlight in both cases severe
modifications of myofibrillar filaments which appear
disorganized with granular rod-like structures, cytoplasmic
bodies and myofibrillar disarray with focal perpendicular
arrangement. The presence of myofibrillar disarray and
granulo-filamentous material is likely to derive from the
Z-line, and this is supported by immunofluorescence for
desmin and myotilin, which are usually observed in myofibrillar myopathies. Among myopathies characterized by
myofibrillar derangement, the myofibrillar myopathies
have been well described.11–14 They are a group of muscle
disorders associated with similar morphologic features
consisting of myofibrillar disorganization originating from
the Z-disk followed by accumulation of myofibrillar degradation products. They may be defined by the presence
of rimmed vacuoles, associated with ectopic expression
of multiple proteins that include desmin, neural cell
adhesion molecule, plectin, gelsolin, ubiquitin, Xin, TAR
DNA-binding protein 43 and cochaperones, including
aB-crystallin, heat shock protein-27.11–14 Myotilinopathy
(LGMD1A) and filaminopathy have been reported as a
subset of myofibrillar myopathy. Both myotilinopathy and
filaminopathy (the so-called Z-disk diseases) exhibit the
morphological findings typical of myofibrillar myopathy
with filament accumulation including Z-disk alterations.12,13
Particularly in filaminopathy, strongly positive for filamin C,
ultrastructural examination revealed major myofibrillar
abnormalities, including accumulation of desmin-positive
granulo-filamentous material. In addition, also large
autophagic vacuoles and mitochondrial aggregates in the
abnormal fiber regions were observed.15–17 The disease
mechanism in filaminopathy is still unclear, but it may
involve structural alterations of the Z-disk caused by dysfunctional proteins or their abnormal accumulation due to
defective degradation.12,13 In particular, among cytoskeletal
proteins, desmin, with its binding partners, forms a three-
dimensional scaffold around Z-disks, thereby interlinking
with myofibrils and nuclei, mitochondria and sarcolemma.
Several studies demonstrated that the filamentous desmin
network plays an essential role in the subcellular positioning and function of mitochondria.11–14 Indeed,mitochondrial
accumulation has been clearly showed in LGMD1F, and it
was confirmed also in the present two cases, where rare
paracrystalline inclusions were found similar to those previously described.4
The contemporary presence of autophagosomes and
several myelin figures is the morphological substrate
of a protein quality-control disturbance related to the
ubiquitin–proteasome system (UPS) and the autophagiclysosomal pathway.11 A recent study showed that desmin
mutants impair the proteolytic function of the UPS and the
autophagic–lysosomal pathway (types 1 and 2 of programmed cell death).11
Electron microscopy is useful in the diagnostic workup
of chronic myopathies identifying pathological protein
aggregation, cytoplasmic/spheroid bodies, and signs of
myofibrillar degeneration, such as sarcoplasmic granulofilamentous material, autophagic vacuoles and myelin-like
whorls.11 To address the alteration of the Z-line, both degeneration, streaming, irregularities and Z-line loss should be
detected. Furthermore, additional features can be found,
such as depletion or accumulation of mitochondria.
Our morphological and ultrastructural data seem to
suggest in our LGMD1F cases a myopathy phenotype
similar to those described for Z-disk diseases.Although the
genetic defect is still under investigation, it is possible to
hypothesize that the mutant protein in LGMD1F might
lead to disarrangement of desmin-associated cytoskeletal
networks.
CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.
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8. Gambelli S, Malandrini A, Ginanneschi F et al. Mitochondrial abnormalities in genetically assessed oculopharyngeal muscular dystrophy. Eur Neurol 2004; 51:
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9. Van Der Sluijs BM, Hoefsloot LH, Padberg GW, Van
Der Maarel SM, Van Engelen BG. Oculopharyngeal
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Vorgerd M, van der Ven PFM, Bruchertseifer V et al. A
Mutation in the dimerization domain of Filamin C
causes a novel type of autosomal dominant myofibrillar myopathy. Am J Hum Genet 2005; 77: 297–304.
Kley RA, Hellenbroich Y, van der Ven PFM et al.
Clinical and morphological phenotype of the filamin
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70
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806
Department of Genetics, Sydney, Australia; 5 Alfred Hospital, Department
of Anatomical Pathology, Melbourne, Australia; 6 University of Western
Australia, Centre for Medical Research, Perth, Australia; 7 University of
Western Australia, Centre for Neuromuscular and Neurological Disorders,
Perth, Australia
The dystrophinopathies are allelic muscular dystrophies caused by Xlinked recessive mutations in dystrophin, with only rare reports of
asymptomatic adult males. The static cognitive impairment seen in dystrophinopathies is thought to be due to altered expression of dystrophin isoforms, but has only once been described in the absence of muscle weakness.
We identified a cohort of patients with unexpected copy number variants
(CNV) in the dystrophin gene, on microarrays performed for
developmental delay or intellectual disability, in whom muscle weakness
was minimal or absent. Subjects with a dystrophin CNV referred to the
neurology or genetics departments at RCH Melbourne or GHSV were
assessed. An additional family was identified from the CHW, and included.
Twelve probands had a CNV in the dystrophin gene on micro-array
testing. Eight (seven male, one female; seven deletions, one duplica-tion),
age 0–9 years, had atypical phenotypes as described above. CNVs were
found in 10 family members (five males and five females), including three
asymptomatic adult males. In all but one family, MLPA confirmed loss of
exons. Muscle weakness was absent or minimal. Serum CK was normal or
mildly elevated. Muscle biopsy revealed morphologically nor-mal muscle
with normal dystrophin immunoreactivity. Microarray testing has revealed
an extended spectrum of clinical phenotypes associated with mutations in
dystrophin, that may include isolated developmental delay and
asymptomatic individuals. Further study is required to understand the
molecular basis of the apparent absence of muscle pathology in these
patients, and the relationship of the dystrophin deletion to cognitive
impairment.
http://dx.doi:10.1016/j.nmd.2012.06.016
D.O.3
Next generation sequencing applications are ready for genetic diagnosis of
muscular dystrophies
M. Savarese 1, A. Torella 1, M. Mutarelli 2, M. Dionisi 2, T. Giugliano 3,
G. Di Fruscio 3, M. Iacomino 3, A. Garofalo 3, S. Aurino 3, F. Del Vecchio
Blanco 3, G. Piluso 3, L. Politano 4, M. Fanin 5, C. Angelini 5, V. Nigro 3
1
Seconda Università degli Studi di Napoli, Laboratorio di Genetica Medica,
Dipartimento di Patologia Generale, Napoli, Italy; 2 TIGEM, Telethon
Institute of Genetic and Medicine, Napoli, Italy; 3 Seconda Università degli
Studi di Napoli, Dipartimento di Patologia Generale, Napoli, Italy; 4 Seconda Università degli Studi di Napoli, Cardiomiologia e Genetica Medica,
Napoli, Italy; 5 Università degli Studi di Padova, Department of Neurosciences, Padova, Italy
Next generation sequencing (NGS) is having a tremendous impact on
our knowledge of different aspects of biology. It can be also very powerful to study patients with heterogeneous genetic conditions, like muscular dystrophies. First, to identify new genes using “exome resequencing”.
Second, to diagnose mutations in all the known causative genes, when
used as targeted approach. Third to obtain a knowledge of the impact
of mutations on mRNA expression and splicing in diseased muscle.
We used NGS to identify new genes by whole exome sequencing. We
sequenced the whole exome of four family members with LGMD1F separated by up to eleven meioses and identified a single shared novel heterozygous frame-shift variant. This causes a nonstop change in the
Transportin 3 (TNPO3) gene that encodes a member of the importin-b
super-family. To reach the second task, we first recruited 160 familial
cases of nonspecific limb-girdle muscular dystrophies with apparent
autosomal inheritance. All DNA samples were first enriched for
486,480 bp, covering 2447 exons of 98 genes by using the Haloplex tech-
nology with the use of barcodes. We the performed pooled NGS of all
samples and identified a number of mutations, then verified by Sanger
sequencing. Cases were also studied by the Agilent MotorChip CGH
array version 3.0 to identify deletions or duplications. Finally, in selected
cases, we performed the RNA-Seq starting from a muscle biopsy sample.
We converted mRNA to cDNA and purified it by a customized SureSelect Target Enrichment System, focused on the same 98 mRNAs. The
probes had a 4 coverage with a total target of 1.41 Mb of sequences/
sample. These cDNAs were sequenced using barcodes trying to obtain
an average sequencing coverage of at least 100. Our results confirm
that there is a very high genetic heterogeneity in muscular dystrophies
and that NGS-based DNA and RNA testing are ready for diagnostic
use.
http://dx.doi:10.1016/j.nmd.2012.06.017
D.O.4
Next generation sequencing for genetic diagnosis and gene identification in
myopathies
J. Bohm 1, N. Vasli 1, U. Schaffer 1, S. Le Gras 2, B. Jost 2, N.B. Romero 3,
N. Levy 4, E. Malfatti 3, V. Biancalana 1, J. Laporte 1
1
IGBMC, Translational Medecine, Illkirch, France; 2 IGBMC, Illkirch,
France; 3 Insitut de Myologie, Unite de Morphologie Neuromusculaire,
Paris, France; 4 Faculté de Médecine de Marseille, Inserm UMRS 910,
Marseille, France
Myopathies are rare diseases with a high impact on patients, fami-lies
and the health care system. Despite tremendous efforts, about half of
patients do not have a molecular diagnosis. This is mainly due to genetic
heterogeneity, the fact that very large genes known to be mutated in
myopathies are difficult to screen, and the presence of yet unidentified
genes. We provide the proof-of-principle that next genera-tion sequencing
(NGS) can be used for molecular diagnosis, to screen large genes, and to
identify novel genes. For molecular diagnosis, we used a custom capture
library to enrich the coding sequence and intron–exon boundaries of 267
genes known to be mutated in neuro-muscular diseases. We could detect all
known mutations in previously characterized patients, including
homozygous, heterozygous, exonic, intronic, point, small indel mutations
and a large deletion. The cost to sequence these 267 genes is lower than to
test one gene by the con-ventional Sanger method. We then tested several
patients without molecular diagnosis and could find mutations in several of
them includ-ing mutations in TTN, the largest human gene. We also used
exome sequencing in different myopathy cohorts and identified diseasecausing mutations in RYR1 and NEB genes, large genes not screened on
rou-tine if RNA is not available. Phenotypes of patients with RYR1 mutations were very heterogeneous, supporting that NGS broadens genotype–
phenotype correlations and represents an unbiased approach to investigate
mutation/gene frequency in myopathies. Next we used exome and genome
sequencing to identify disease-causing genes in spe-cific myopathies for
which no causative genes were previously known. We found mutations
either in genes previously linked to other myopa-thies or in novel genes.
Examples will be presented. Next generation sequencing will accelerate
mutation discovery for the benefit of patient diagnosis and a better
understanding of muscle function under normal and pathological
conditions.
http://dx.doi:10.1016/j.nmd.2012.06.018
D.O.5
A combination of linkage analysis and exome sequencing identifies a new
gene for X-linked Charcot–Marie–Tooth neuropathy
71
June 12th, 2012
Muscle disorders
Tuesday, June 12, 2012, 11:30 - 12:30
New quantitative MRI indexes useful to investigate muscle diseases
C. Angelini, M. Fanin, E. Peterle (Padova, IT)
Objectives: We propose new types of quantitative measurement to evaluate muscle atrophy: the quadriceps index (QI)
and the left vastus lateralis index (VLI), measuring by MRI their area.
Methods. We have used T1 sequences on thigh muscle MRI, at about 15 cm from the head of the femur (second slide of
MRI in lower extremities). In these sequences we measured the muscle area of the left quadriceps femoris and of the left
vastus lateralis. These measurement were carried out in 11 patients with various types of myopathies i.e. two cases of
lipid storage myopathies, 1 amyotrophic lateral sclerosis, 1 facio-scapulo-humeral dystrophy, 1 myofibrillar myopathy, 1
metabolic myopathy, 2 patients with LGMD2A, 1 patient with LGMD1F, 1 localized myositis ossificans, 1 aspecific
myopathy. Muscle biopsies of these patients were further investigated by morphometry and molecular markers of atrophy
or autophagy i.e.MURF, LC3.
Results: We performed the measurement of muscle area of quadriceps femoris (Q.I) in 11 patients, that resulted in
average 3711 mm2 ± SD 792. In this group of patients we have identified two subgroups, one including 5 patients with a
high degree of muscle atrophy (highly atrophic group), whose values ranged from 2400 to 3400 mm2 (mean 2966), and
one including 6 patients with a low degree of atrophy (low atrophic group), whose values ranged from 3700 to 5000 mm2
(mean 4332).
The measurement of muscle area of vastus lateralis in 11 patients was in average 963 mm2 ± 303. In the atrophic subgroup the values ranged from 400 to 900 mm2 (mean 658.7), while in the normal sub-group the values ranged from 900
to 1400 mm2 (mean 1217.8).
Conclusion: Both the quadriceps and the vastus lateralis indexes appear useful to evaluate muscle atrophy in LGMDs,
ALS and metabolic myopathies: a high degree of atrophy of QI was found in calpainopathy, motor neuron disease and
Limb Girdle Muscular Dystrophy type 1F, the measurement of the VLM appeared less specific since it includes a larger
area. Both these quantitative indexes obtained by muscle MRI, could be used as clinical outcomes of treatment in
neuromuscular disorders in order to follow up and study natural history or the effect of various type of treatments
(steroids, carnitine, etc.). A promising field of investigation appears the correlation of imaging indexes with other atrophy
parameters obtained in muscle biopsy, i.e with the cross sectional area or fibers or with molecular markers of atrophy
and autophagy.
72
2012
P115
IDENTIFICAZIONE DI NUOVI GENI COINVOLTI NELLE DISTROFIE MUSCOLARI DEI CINGOLI MEDIANTE ARRAYS
E SEQUENZIAMENTO DI NUOVA GENERAZIONE (NGS)
1
3
2
3
3
2
1
A. Torella , F. Del Vecchio Blanco , M. Dionisi , A. Garofalo , M. Iacomino , M. Mutarelli , M. Savarese , G. Piluso
1
1
, V. Nigro
Dip. di Patologia Generale-Lab. di Genetica Medica, Seconda Università degli Studi di Napoli, Telethon Institute of
Genetics and Medicine (TIGEM)
2
Telethon Institute of Genetics and Medicine (TIGEM)
3
Dip. di Patologia Generale-Lab. di Genetica Medica, Seconda Università degli Studi di Napoli
1
Campioni di DNA di soggetti affetti da distrofia muscolare sono stati da noi analizzati per i geni responsabili di LGMD:
il 30% dei pazienti presentava una mutazione nel gene CAPN3, il 10% nel gene DYSF , il 10% nei geni dei sarcoglicani
e un altro 10% negli altri geni noti LGMD:LGMD2A-N. Una significativa percentuale di pazienti con LGMD non aveva
alcuna mutazione nei 18 geni LGMD scoperti finora. In particolare, il 40% dei pazienti non ha una diagnosi molecolare.
La spiegazione è da ricercare nell'elevata eterogeneita' genetica. Le tecniche tradizionali presentano l'inconveniente di
concentrare la ricerca delle mutazioni su un singolo gene alla volta. Inoltre, gli attuali esami genetici sono lunghi, costosi e
senza effetti. I nuovi potenti approcci per lʼanalisi del DNA, come la next-generation sequencing (NGS) sono in procinto di
rivoluzionare il campo con un singolo strumento in grado di analizzare lʼintero genoma umano per molte volte.
La nostra ricerca ha combinato analisi di linkage basata su SNP array e la tecnologia NGS al fine di scoprire mutazioni
“orfane” di LGMD.
I pazienti oggetto di studio sono stati selezionati secondo i seguenti criteri: a)diagnosi clinica di LGMD; b)diagnosi molecolari
non concluse, c)la maggior severità della malattia;
Tutti gli altri casi sono stati studiati mediante 8x60k Motor Chip, un array-CGH basato su oligonucleotidi con una copertura esonica completa dei geni coinvolti nelle malattie neuromuscolari che permette di individuare delezioni o duplicazioni
deleterie. Gli esomi di 16 soggetti appartenenti a 7 diverse famiglie sono stati sequenziati mediante NGS utilizzando la
piattaforma SOLID e, in parallelo, (1 famiglia) lʼ Illumina HiSeq2000. Un certo numero di mutazioni sono state identificate.
In particolare quattro membri affetti di una famiglia con ereditarietà AD (LGMD1F) presentano una singola mutazione (frame-shift) non trovata negli altri membri della famiglia o controlli. In una seconda famiglia LGMD con ereditarietà AR abbiamo recentemente identificato una mutazione missenso in omozigosi nel gene ACADVL che è condivisa da tutti i membri
affetti della famiglia e da altri pazienti provenienti dalla stessa area geografica.
73
October 30th, 2011
Acta Myologica • 2011; XXX: p. 168
ADDENDUM
Proceedings of the XI Congress
of the Italian Association
of Myology
Cagliari, May 2011
LGMD 1(F) - A pathogenetic hypothesis
based on histopathology and
ultrastructure
G. Cenacchi, E. Peterle, L. Tarantino, V. Papa, M. Fanin,
C. Angelini
Clinic Department of Radiologic and histopathologic Sciences,
University of Bologna, Department of Neurosciences and
VIMMM, University of Padua
A large Spanish kindred with apparently autosomal dominant inheritance has been reported with proximal limb and axial
muscle weakness. Clinical, histological and genetic features
have been described in 5/32 patients. A novel LGMD disease
locus at chromosome 7q32.1-32.2 has been identified, but any
defects were detected in filamin C, a gene candidate from this
chromosomal region encoding actin binding protein highly expressed in muscle. We report a clinico-pathological study of two
patients (mother and daughter) from the same Spanish family.
Age at onset was in the teens: earlier onset in the daughter with
a faster weakness progress confirms an apparent genetic anticipation. Morphologic findings were similar in both cases: H&E
notices increased fiber size variability, fiber atrophy, endo- and
perimisial connective tissue, and acid-phosphatase positive vacuoles. The ultrastructural study confirmed fiber atrophy, abnormal mitochondria accumulations and autophagosomal vacuoles
containing cell debris and pseudomyelin figures: no filamentous
inclusions were detected which are usually associated with a
HIBM. Many alterations of myofibrillar component were easily detected such as prominent disarray, rod-like structures with
granular aspect, and occasionally cytoplasmic bodies. Our morphological data support the hypothesis that other actin-encoding
proteins such as FSCN3, and KIAA0265 from the same critical region may represent attractive candidate genes in the LGMD 1(F) pathogenetic mechanism.
Abstract omitted in Acta Myologica, Vol. XXX, June 2011
168
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75
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77
August 8th, 2000
Autosomal dominant limb-girdle
muscular dystrophy
A large kindred with evidence for anticipation
J. Gamez, MD; C. Navarro, MD; A.L. Andreu, MD; J.M. Fernandez, MD; L. Palenzuela, MS; S. Tejeira, MS;
R. Fernandez–Hojas, MS; S. Schwartz, MD, PhD; C. Karadimas, PhD; S. DiMauro, MD; M. Hirano, MD;
and C. Cervera, MD
Article abstract—Background: Fourteen genetically distinct forms of limb-girdle muscular dystrophy (LGMD) have been
identified, including five types of autosomal dominant LGMD (AD-LGMD). Objective: To describe clinical, histologic, and
genetic features of a large Spanish kindred with LGMD and apparent autosomal dominant inheritance spanning five
generations. Method: The authors examined 61 members of the family; muscle biopsies were performed on five patients.
Linkage analysis assessed chromosomal loci associated with other forms of AD-LGMD. Results: A total of 32 individuals
had weakness of the pelvic and shoulder girdles. Severity appeared to worsen in successive generations. Muscle biopsy
findings were nonspecific and compatible with MD. Linkage analysis to chromosomes 5q31, 1q11-q21, 3p25, 6q23, and 7q
demonstrated that this disease is not allelic to LGMD forms 1A, 1B, 1C, 1D, and 1E. Conclusions: This family has a genetically
distinct form of AD-LGMD. The authors are currently performing a genome-wide scan to identify the disease locus.
NEUROLOGY 2001;56:450–454
The limb-girdle muscular dystrophies (LGMD) comprise a genetically diverse group of muscle disorders
with predominantly proximal limb and axial muscle
weakness. Because of molecular genetic discoveries
and improved clinical criteria, the classification and
nomenclature of LGMD have evolved over the last
decade.1,2 Many LGMD disorders are autosomal recessive traits, but at least five well-characterized
forms have been reported in recent years.3-12
We studied a large Spanish kindred with 32 affected individuals and apparently autosomal dominant inheritance spanning five generations. Here, we
describe the clinical phenotype and morphologic findings in five patients who underwent muscle biopsy,
and preliminary genetic investigations of this new
type of autosomal dominant LGMD (AD-LGMD).
Patients and methods. A total of 61 individuals from
five generations of a family from eastern Spain were examined. Serum creatine kinase (CK), aspartate aminotransferase (ASAT), and alanine aminotransferase (ALAT)
determinations were performed on all subjects. Twelve underwent neurophysiologic examinations, and five had
muscle biopsy. Other investigations included electrocardiography (ECG) in 12 patients, echocardiography in six
patients, and MRI of the brain in two patients.
Subjects were considered affected when clinical exami-
nation revealed a characteristic pattern of muscular weakness, primarily affecting the pelvic and shoulder girdles.
Muscle strength was assessed using the British Medical
Research Council (MRC) Scale; 26 muscle groups were examined bilaterally. Functional ability was measured according to the scales designed by Vignos and Brooke.13-14
Age at onset was determined using a standardized clinical
questionnaire form asking clinically affected individuals to
identify their first symptoms from a list that included a
waddling gait and difficulty in climbing stairs, raising
hands above the head, lifting, running, and rising from a
chair or squatting position. A total of 32 family members
were clinically affected. In figure 1, individuals are identified
by generation number (Roman numerals) followed by birth
order position, reading from left to right (Arabic numerals).
Skeletal muscle biopsy specimens from the deltoid or
vastus lateralis were oriented, snap-frozen in liquid
nitrogen-chilled isopentane and the cryostat-cut sections
were stained using standard histochemical methods. Immunohistochemistry was performed using the following
antibodies: desmin and vimentin (Biogenex, CA), ubiquitin
(DAKO, DK), Tau protein and Beta-amyloid (Sigma, MO),
and dystrophin, sarcoglycans, and the amino terminal of
utrophin (DRP2, Novocastra Laboratories, Newcastle upon
Tyne, UK). A small portion of each sample was fixed in
glutaraldehyde and processed for ultrastructural
examination.
Genetic linkage studies were performed to exclude chro-
From the Department of Neurology (Drs. Gamez and Cervera) and Centre d’ Investigacions en Bioquimica i Biologia Molecular (Drs. Andreu and Schwartz,
and L. Palenzuela), Hospital Vall d’ Hebron, Barcelona; Department of Pathology and Neuropathology (Dr. Navarro, S. Tejeira, and R. Fernandez–Hojas)
Hospital do Meixoeiro; Department of Clinical Neurophysiology (Dr. Fernandez), Hospital Xeral-Cies, Vigo, Spain; and H. Houston Merritt Clinical Research
Center for Muscular Dystrophy and Related Diseases (Drs. Karadimas, DiMauro, and Hirano), Department of Neurology, Columbia University College of
Physicians and Surgeons, New York.
Supported by the Spanish Fondo de Investigación Sanitaria (FIS 00/797).
Received June 8, 2000. Accepted in final form October 27, 2000.
Address correspondence and reprint requests to Dr. Josep Gamez, Department of Neurology, Hospital Gral, Vall d’Hebron, Passeig Vall d’Hebron, 119-125,
08035 Barcelona, Spain; e-mail: [email protected]
450 Copyright © 2001 by AAN Enterprises, Inc.
78
August 8th, 2000
Figure 1. Drawing of the pedigree. Clinically affected members are shown in black. Roman numerals indicate generation
number, and Arabic numerals birth order position within the generation.
mosomal loci associated with other forms of AD-LGMD
(LGMD1), hereditary inclusion body myopathy (HIBM),
and Bethlem myopathy.
Blood samples were taken from all 61 members of the
pedigree after informed consent. After DNA extraction
from the blood buffy coats, a set of fluorescent-labeled microsatellite simple sequence repeat (SSR) markers spanning the five known loci of LGMD1 were genotyped using
an ABI Prism 310 Genetic Analyzer (Perkin Elmer, Foster
City, CA).
In addition, SSR markers encompassing the loci of autosomal recessive HIBM (AR-HIBM),15 autosomal dominant
HIBM (AD-HIBM),16 Bethlem myopathy,17-18 and facioscapulohumeral dystrophy (FSHD)19 were genotyped.
These SSR markers were as follows:
1. LGMD 1A (5q31): D5S410, D5S436, D5S2115, and
D5S471
2. LGMD 1B (1q11-q21): D1S218, D1S196, D1S2878,
D1S484, D1S2635, D1S498, D1S252, and D1S2726
3. LGMD 1C (3p25): D3S1277, D3S1266, D3S2338,
D3S1263, D3S1304, and D3S1297
4. LGMD 1D (6q23): D6S308, D6S292, D6S262, and
D6S287
5. LGMD 1E (7q): D7S427, D7S2465, D7S798, D7S636,
and D7S661
6. AR-HIBM (9p1-q1): D9S161, D9S1817, D9S273, and
D9S175
7. AD-HIBM (17p13.1): D17S938, D17S1852, D17S799,
and D17S921
8. Bethlem myopathy (2q37 and 21q22.3); D2S206,
D2S338, D2S125, D21S1252, D21S1255, and D21S266
9. FSHD (4q35): D4S2920, D4S1535, D4S2924, and
D4S426
Two-point (marker-to-disease) analysis was performed
with the MLINK option of FASTLINK 4.0.20
The disease was considered autosomal dominant with
90% penetrance. The disease gene frequency was estimated at 1/100,000. Marker allele frequencies were estimated by allele counting of all genotyped subjects. The
LINKMAP option was used for three-point analyses.
Marker order and intermarker distances were based on
the Genethon linkage map.
Results. Characteristic muscle weakness predominantly
involving the pelvic and shoulder girdle proximal muscles
was shown in 32 individuals (15 men, 17 women) between
the ages of 7 and 66 years (mean 34.5, SD 14.4 years).
Age at onset ranged from less than 1 to 58 years (mean
16.3, SD 15.5 years). Two groups were delineated based
on age at onset: a juvenile form, with onset before age 15
(65.6% of patients) and an adult-onset form, starting
around the third or fourth decade (28.0%).
Symptoms of pelvic girdle muscular weakness were
noted at onset in 81.1% of cases. Commonly affected muscles were the iliopsoas (96.0%), gluteal (75.0%), hip adductors (71.8%), deltoid (90.6%), biceps brachii (68.7%),
paraspinal (65.6%), and neck flexors (62.5%). Pelvic girdle
impairment was more severe and occurred earlier than in
the shoulder girdle. Proximal muscle weakness ranged
from MRC grade 0 to 4/5, with symmetric distribution.
Distal weakness appeared late in the disease’s course or
accompanied initial presentation in severely affected
juvenile-onset patients, frequently affecting the extensor
digitorum, tibialis anterior, and toe extensor muscles.
Six patients had scapular winging. Two juvenile-onset
patients showed mild facial weakness 10 years after onset.
Early-onset patients had generalized muscular wasting, predominantly involving the quadriceps, gluteus, deltoid, biceps,
infraspinatus, and supraspinatus muscles (figure 2).
Early joint contractures were not present. Three individuals developed Achilles tendon contractures late in the
disease’s course. Four subjects showed scoliosis or hyperlordosis. All belonged to the juvenile-onset group.
Respiratory muscles were clinically affected in four patients with juvenile-onset form. Mean forced vital capacity
in these patients was 38.6% of predicted values.
No patient had ptosis, ophthalmoparesis, dysphagia,
speech disturbances, calf hypertrophy, myalgia, or intellectual deterioration.
Weakness progress during the 8-year follow-up showed
two patterns: relatively slow in adult-onset subjects, and
faster in juvenile-onset subjects. The progression rate
seemed linear. Two patients with onset before age 14 became wheelchair-bound before age 28. A boy with onset at
age 1 year needed assistance walking by age 12, and his
February (2 of 2) 2001 NEUROLOGY 56 451
79
August 8th, 2000
Figure 2. Patient showing lordosis, scapular winging,
proximal wasting affecting the pelvic and shoulder muscles, and a sparing of the facial muscles.
mother’s help with most everyday tasks (Vignos’ scale
grade 6).
Mean CK was 589, ranging between 47 and 2,920 (normal 150 U/L). Normal CK was recorded in 40.6% of patients. Mean ASAT and ALAT were 35.7 and 44.5 U/L.
Electromyography (EMG) showed myopathic changes
with short duration, polyphasia, and low-amplitude poten-
tials, which were more pronounced in the proximal muscles. Sensory and motor nerve conduction velocities were
normal.
Brain MRI, ECG, and echocardiograms, when performed, were normal.
The inheritance pattern is consistent with autosomal
dominant transmission; 44 of 76 (58%) children of affected
parents manifested the disease. We identified 26 affected
parent/affected child pairs. The pair-wise data comprises
18 pairs with a generation III parent and eight pairs with
a generation IV parent. In the generation III parent pairs,
we observed a mean decrease of 28.5 years in an offspring’s
onset age. In the generation IV parent pairs, the mean
decrease in an offspring’s onset age was 13.2 years. Thus,
apparent anticipation was significant (p 0.000 in generation III parents and p 0.002 in generation IV parents).
Overall comparison of age at onset curves in life table
analysis between patients of generations III, IV, and V
showed a decrease in age at onset (Wilcoxon test statistic
16.84, p 0.0002). We also examined the effects of
parental gender origin on anticipation. The mean difference in age at onset between parent and child in 17 mother/child pairs was 1.6 years younger than in 9 father/child
pairs (two-tailed Mann–Whitney U test 59.5, p 0.367).
In our data, no evidence suggests a significant effect of
parental gender on age at onset.
Muscle biopsy. Light microscopy. Open muscle biopsy was performed on patients III-8, IV-6, IV-11, IV-21,
and V-11. When biopsied, their ages varied between 9 and
59 years (mean 31.4 years). Morphologic findings were
similar in all cases, composed of abnormal fiber size and
shape variation, increased endo- and perimysial connective
tissue, scattered degenerative fibers with myophagia, abnormal Z-bands, and, in three of five cases, prominent
rimmed vacuoles (figure 3A). Central nuclei were occasionally present. Fiber type differentiation and distribution
were normal. One patient (V-11) showed a significant
number of ragged-red fibers (RRF) (15%); cytochrome c
oxidase (COX)–succinate dehydrogenase technique disclosed between 5 and 30% COX-negative fibers in three of
five cases. All RRF were COX-negative, but COX-negative
fibers without signs of mitochondrial proliferation were
also present. Immunohistochemical stains for dystrophin
and sarcoglycans were normal. No abnormal deposits of
tau, ubiquitin, or -amyloid proteins were found. Desmin
was overexpressed in some fibers, but was not abundant
enough for consideration as a significantly abnormal
desmin accumulation. Vimentin and dystrophin-related
protein overexpression in scattered small fibers without
Figure 3. (A) Muscle cryostat section in
Subject IV-6. Notice increased fiber size
variability, one small fiber with two
prominent rimmed vacuoles (bottom
right corner) and a fiber with subsarcolemmal basophilia and marked intermyofibrillary network, indicative of
mitochondrial proliferation (center) (hematoxylin– eosin 60 before reduction).
(B) Electron micrograph in Subject
V-11. Skeletal muscle fiber cut transversally. Notice increased number of paranuclear mitochondria with abnormal cristae and paracrystalline inclusions
(8,000 before reduction).
452 NEUROLOGY 56 February (2 of 2) 2001
80
August 8th, 2000
dystrophin deficiency was considered evidence of
regeneration.
Electron microscopy. Three cases showed autophagic
vacuoles with prominent pseudomyelin figures and dense
bodies. One case (IV-6) showed intracytoplasmic bundles of
16- to 18-nm filaments. Despite thorough searching, no
additional filamentous inclusions were observed in the nuclei or in the other four patients’ cytoplasm or nuclei. One
patient (V-11) showed prominent subsarcolemmal and
paranuclear abnormal mitochondria accumulations, many
with paracrystalline inclusions (see figure 3B).
Linkage analysis. Using genotype data of SSR markers, three-point analyses excluded linkage of the LGMD in
our pedigree to known chromosomal loci for the following:
LGMD forms 1A, 1B, 1C, 1D, and 1E, autosomal dominant
and recessive HIBM, Bethlem myopathy, autosomal dominant Emery–Dreifuss dystrophy (AD-EDMD), and FSHD
based on three-point lod scores 2.0.
Discussion. Thirty-two patients were identified
from a large Spanish family with autosomal dominant muscular dystrophy. The disorder fulfills
LGMD diagnostic criteria,21 with the following clinical features: slowly progressive proximal symmetric
weakness with predominantly lower limb onset, normal to mildly raised CK activity, myopathic EMG
and muscle biopsy changes, and normal skeletal
muscle dystrophy staining.
Variability was observed in age at onset, muscular
symptomatology, and progression rates, with two patient groups differentiated by severity and progression: an adult-onset form, around the fourth decade,
and a juvenile form, beginning before age 15 years.
Patients with severe functional disability belonged to
the second group.
Dysarthria, cardiac involvement, calf hypertrophy, and contractures are features of other ADLGMD forms, not seen in our patients. Another
feature of our family was an early onset age (mean
16.3 years). In nearly two thirds of patients, onset
occurred in childhood or adolescence. Patients with
onset before 12 years of age presented with generalized muscular weakness.
We noted an anticipation phenomenon in 26 parent–
child pairs, with the parents two and three generations removed from a common ancestor. Earlier
onset in children than in parents suggested genetic
anticipation. Disease severity seems unrelated to the
transmitting parent’s gender. Anticipation phenomenon was described in LGMD-1.6
The absence of facial, bulbar, or cardiac impairment excluded other myopathies. The lack of early
joint contractures suggests our AD-LGMD pedigree
differs from Bethlem myopathy or AD-EDMD. We
excluded linkage to chromosomal loci for Bethlem
myopathy and AD-EDMD.17,18,22-24
Some patients with FSHD lack facial involvement,
thus resembling LGMD. However, in our family the
absence of shoulder girdle muscle weakness asymmetry, predominant muscle weakness of scapular
fixators, hearing loss, and facial weakness affecting
eye closure and perioral muscles makes FSHD un-
likely. Onset before 5 years of age is rare in FSHD,
but was observed in our pedigree. We excluded linkage of the diseases to the 4q35 FSHD locus.19
Muscle biopsies showed rimmed vacuoles in three
patients. Initially, a diagnosis of HIBM was considered because of this and the presence of filamentous
inclusions.25
Most HIBM cases reported have shown autosomal
recessive inheritance26-27 but AD-HIBM has been
rarely described.28-29
However, rimmed vacuoles without the characteristic filaments are frequent nonspecific findings in
different muscle disorders, including forms of
LGMD17,30 and primary dystrophinopathies.31 We excluded linkage to the identified loci for the identified
loci of autosomal recessive and dominant HIBM.15-16
We interpreted the mitochondrial proliferation in
one patient’s muscle as a secondary phenomenon.
Five AD-LGMD (LGMD1) forms have so far been
delineated.2,5,8-12 Types 1B and 1E are associated
with cardiologic abnormalities, including atrioventricular conduction disturbances, arrhythmias and
sudden death. Type 1A is characterized by a dysarthric speech pattern not observed in our patients.
LGMD 1C is caused by caveolin-3 mutations.9-10
LGMD 1D has been mapped to chromosome 6q23.11,12
Our linkage-analysis data using markers for those
loci found no association, suggesting a genetically
distinct disorder. We are performing a genome-wide
scan to identify the disease locus.
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82
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