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ISTITUTO LOMBARDO - ACCADEMIA di SCIENZE e LETTERE ISSN 2279-5251

INCONTRO DI STUDIO N. 66

DISTROFIE MIOTONICHE: malattie genetiche da RNA tossico

INCONTRO DI STUDIO - DISTROFIE MIOTONICHE: MALATTIE GENETICHE DA RNA TOSSICO

Milano, 28 giugno 2012

A cura di Carlo Pellicciari

Istituto Lombardo di Scienze e Lettere MILANO 2013


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ISTITUTO LOMBARDO - ACCADEMIA di SCIENZE e LETTERE ISSN 2279-5251

INCONTRO DI STUDIO N. 66

DISTROFIE MIOTONICHE: malattie genetiche da RNA tossico

Milano, 28 giugno 2012

A cura di Carlo Pellicciari

Istituto Lombardo di Scienze e Lettere MILANO 2013


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Comitato Scientifico: FIORENZA DE BERNARDI PAOLO MAZZARELLO CARLO PELLICCIARI

Pubblicato con il contributo di: Università degli Studi di Milano Università degli Studi di Pavia Fondazione Malattie Miotoniche


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SALUTO DEL PRESIDENTE DELL’ISTITUTO LOMBARDO GIANPIERO SIRONI

Mi è molto gradito dare il benvenuto dell’Istituto Lombardo ai relatori di questo Incontro di studio e a tutti i partecipanti. Il tema dell’Incontro di studio odierno riguarda un gruppo di patologie di grande interesse, non solo da un punto di vista umano, come molte patologie di rilievo, in particolare a matrice ereditaria; ma dal punto di vista scientifico, anche perché in relazione a queste patologie vengono continuamente acquisite nuove conoscenze. Di queste ci parleranno appunto i relatori. Il testo di presentazione dell’Incontro, prima ancora dei riassunti delle relazioni, che sono stati resi disponibili, rivela che lo studio di queste patologie, la comprensione delle cause che ne sono all’origine e della dinamica della loro genesi hanno coinvolto e tuttora coinvolgono diverse discipline, come ha osservato anche Giorgio Semenza, inviando un suo messaggio di adesione: - la patologia medica: va da sé, non occorre spiegarne il motivo; - la genetica: si tratta infatti di malattie geneticamente determinate; - la biologia molecolare: basti dire che l’analisi degli acidi nucleici ha fornito importanti chiarimenti; - la biochimica: menziono in particolare, ma non solo, il coinvolgimento di particolari proteine; - la biologia cellulare: per la compromissione della funzionalità cellulare che consegue alle alterazioni intracellulari; - la stessa bioinformatica: per l’importanza dell’analisi e confronto delle sequenze nucleotidiche; - e forse ne ho dimenticate altre, che pure sono rilevanti in questo ambito.


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Vorrete scusare il fatto che io abbia fatto menzione del contributo di discipline diverse allo studio delle patologie oggetto dell’Incontro odierno, e quindi della sua natura interdisciplinare; mi sembra un caso emblematico del concorso di diversi specialisti per procedere alla conoscenza di problemi complessi; e l’ho fatto perché mi pare del tutto appropriato che l’odierno Incontro di studio si tenga nell’ambito dell’Istituto Lombardo, che vuole avere tra le sue caratteristiche e finalità quella di coinvolgere cultori e specialisti di diverse discipline nel rendere noti i risultati delle ricerche. Debbo dare comunicazione di alcune modifiche al programma. Infatti il prof. Carlo Pellicciari e la prof.ssa Manuela Malatesta non possono essere presenti oggi, a motivo di seri impedimenti sopraggiunti molto recentemente. Il programma previsto verrà, tuttavia, svolto in ogni caso: - l’Introduzione, che avrebbe dovuto essere svolta dal prof. Pellicciari, verrà tenuta dal prof. Giovanni Meola; - la relazione su “Alterazioni nucleari nelle distrofie miotoniche” di Manuela Malatesta e Marzia Giagnacovo, che si prevedeva venisse tenuta da Manuela Malatesta, verrà invece svolta da Marzia Giagnacovo. Immutato il resto. Ho ricevuto diverse lettere di adesione. Tra quelle pervenute, desidero menzionare quella da parte del’Avv. Giuliano Pisapia, Sindaco del Comune di Milano, che ospita il nostro Istituto nella sede di Palazzo Landriani. Desidero ora ringraziare i relatori per aver accettato il nostro invito e gli organizzatori dell’Incontro: Fiorenza De Bernardi, Paolo Mazzarello e Carlo Pellicciari. Torno a rivolgervi il mio saluto e auguro a tutti buon lavoro.


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MYOTONIC DYSTROPHIES: GENETICALLY-BASED DISEASES DUE TO TOXIC RNA CARLO PELLICCIARI (*)

RIASSUNTO. – Le distrofie miotoniche (DM, le forme più diffuse di distrofia muscolare, dopo la distrofia di Duchenne) sono malattie degenerative a base genetica, che mostrano caratteristiche cliniche assai variabili e sono contraddistinte da miotonia (cioè da una prolungata contrazione della muscolatura scheletrica dopo breve stimolazione) e da rallentato rilassamento muscolare dopo contrazione volontaria. Ne esistono due forme: la più grave, detta DM1 (o malattia di Steinert) a la più leggera, DM2. E’ ampiamente accettato che la patogenesi delle DM dipenda dall’accumulo intranucleare di sequenze di RNA espanse, che esercitano un’azione tossica sulla funzionalità cellulare, in dipendenza del sequestro, in localizzazioni nucleari ectopiche, di fattori proteici essenziali alla maturazione dei trascritti. Scopo di questo mini-simposio è descrivere le caratteristiche genetiche e cellulari delle DM, e di dimostrare come la ricerca di base possa fornire indicazioni significative per la diagnosi e la terapia. *** ABSTRACT. – Myotonic dystrophies (DMs, the second most diffuse forms of muscular dystrophy, after Duchenne dystrophy) are genetically-based degenerative neuromuscular diseases exhibiting widely variable clinical features and characterized by myotonia (i.e., a prolonged contraction of skeletal muscles after short stimulation) and a delayed muscle relaxation after voluntary contraction. There are two form of DMs: the more severe DM1 (or Steinert’s disease), and the milder form DM2. The intranuclear accumulation of expanded RNAs is considered as the pathogenetic factor of DMs: the presence of these RNAs exerts a toxic action on cell function which essentially depends on the ectopic sequestration of nuclear protein factors involved in the processing of transcripts. The aim of this mini-symposium is to describe the genetic and cellular bases of DMs, showing how the results of basic research may provide important clues for both diagnosis and therapy.

(*)

Dipartimento di Biologia e Biotecnologie “Lazzaro Spallanzani”, Laboratorio di Biologia Cellulare e Neurobiologia, Università degli Studi di Pavia, Pavia, Italy E-mail: pelli@unipv.it


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Myotonic dystrophies (DMs) are genetically-based degenerative neuromuscular diseases exhibiting widely variable clinical features; they may arise at any age, and have a slowly progressive course. DMs are characterized by myotonia (i.e., a prolonged contraction of skeletal muscles after short stimulation) and by a delayed muscle relaxation after voluntary contraction. They are the second most diffuse forms of muscular dystrophy, after Duchenne dystrophy, with a prevalence 1:8.000. It is estimated in Italy about 8-10.000 patients affected by DM. There are two form of DMs: DM1 (or Steinert’s disease) is more severe and is caused by an unstable expansion of CTG nucleotide triplets in the 3’ non-coding region of the DMPK gene; DM2 is the milder form, and depends on the expansion of the CCTG tetraplet in the first intron of the CNP (ZNF9) gene. It is widely accepted that the pathogenesis of DMs is due to the intranuclear accumulation of expanded RNAs whose toxic action essentially depends on the ectopic sequestration of protein factors involved in the processing of transcripts: as a consequence, the whole function of muscle cells is affected. The aim of this mini-symposium is to describe the genetic and cellular bases of DMs and to demonstrate how the results of the scientific research may provide important clues for both diagnosis and therapy. The invited speakers are well known scientists, active in the field of dystrophy and more generally of skeletal muscle cell biology. Denis Furling is a CNRS researcher at the Institut de Myologie, in Paris. He completed his PhD at the Laval University of Québec (Canada) in 1997, where he followed his post-doctorate with the neurologist Professor Jack Puymirat, at the Faculty of Medicine: here he started his research on myotonic dystrophy which he then continued after his return to France in 2000. Denis Furling has been especially working on the alterations in the myogenic program provoked by CTG expansions, searching for the molecular mechanisms involved in these modifications: to this aim, he developed in vivo and in vitro models reproducing some of the molecular anomalies of DMs. He is presently interested in the reduced proliferative capacity of myogenic precursor cells in severe congenital form of DM1, and his experimental results indicate that defects in the behaviour of satellite cells could be involved in the progressive distal muscle atrophy present in the adult form of the DM1 disease.


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Manuela Malatesta is a researcher at the University of Verona. She has been assistant at the Centre de Microscopie Electronique of the University of Lausanne (Switzerland) where she specialized in electron microscopy and ultrastructural cytochemistry. By combining morphological, histochemical and immunohistochemical analyses at light and electron microscopy she focussed her studies on the structural and functional alterations of RNA transcription and processing in different tissues of aged rodents, including skeletal muscle. She is also involved in a concerted research on the cellular basis of DMs: using microscopical, histochemical and molecular approaches ex vivo and in vitro she demonstrated that both mutations lead to the ectopic nuclear sequestration of several splicing factors causing alteration of messenger RNA processing which is in turn responsible for the multisystemic clinical features typical of these pathologies. Moreover, she found that myotonic dystrophy and sarcopenia share several cell nuclear mechanisms responsible for nuclear dysfunction, opening interesting perspectives for the research of the common bases of skeletal muscle wasting. Marzia Giagnacovo is a PhD student at the Department of Biology and Biotechnology of the University of Pavia, where she is working on the histological, histochemical and molecular features of skeletal muscle cells in aging mice and in human subjects affected by myotonic dystrophy. Giovanni Meola is full professor of Neurology at the University of Milan, and Director of the Department and U.O.C. of Neurology/Stroke Unit at the IRCCS Policlinico San Donato. Pupil of the late lamented Professor Guglielmo Scarlato, he specialized in Neurology at the University of Newcastle Upon Tyne (UK), the Columbia University in New York (USA), and the Universities of Montreal and Quebec (Canada). Since 1995, Giovanni Meola is visiting Professor at the Department of Neurology, University of Rochester, N.Y. (USA) and since 2009 at the Department of Neurology, University of Beograd (Serbia).Over the last thirty years, he performed basic and clinical studies on neuromuscular diseases, with special attention to DMs. With the aim to promote researches on these pathologies and provide support to young researchers in this field, he founded in 2004 the Centre for the Study of Neuromuscular Diseases (CMN; http://www.associazionecmn.com/) and very recently, in December 2011, the Foundation for Myotonic Diseases (FMM; http://www.fondazionemalattiemiotoniche.org/). The FMM, recog-


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nized by the Regione Lombardia, strongly supports basic and clinical researches in the field of myotonic dystrophies. In addition, the Foundation also supports meeting, like the present one organized by Istituto Lombardo Accademia di Scienze e Lettere, to improve knowledge in the field of myotonic disorders, particularly DMs.


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SPLICING ABNORMALITIES IN MYOTONIC DYSTROPHIES DENIS FURLING (*)

RIASSUNTO. – La distrofia miotonica di tipo 1 (DM1) è tra le più comuni forme di distrofia muscolare nell’adulto, caratterizzata da progressivo deperimento e debolezza muscolare, miotonia, difetti di conduzione a livello cardiaco, alterate funzioni cognitive e da diversi altri sintomi multisistemici. La DM1 è una malattia ereditaria autosomica dominante, causata da un’instabile espansione (da ~50 a più di 1.000 ripetizioni) della tripletta nucleotidica CTG nella regione non-codificante all’estremità 3’ del gene DMPK. L’espressione di RNA per DMPK contenenti l’espansione CUG supporta l’ipotesi di una effetto tossico dell’RNA per “acquisizione di funzione”, come meccanismo alla base del fenotipo distrofico. Un meccanismo simile è pure coinvolto nella eziopatologia della distrofia miotonica di tipo 2 (DM2), che ha aspetti clinici comuni alla DM1 ed è causata dall’espansione della sequenza CCTG nel primo introne del gene CNP (ZNF9). In entrambe le distrofie miotoniche, l’accumulo a livello del nucleo cellulare di RNA contenenti le sequenze CUG/CCUG espanse altera l’attività di fattori proteici (quali MBNL1 e CUG-BP1) che legano gli RNA nucleari, con conseguente sregolazione dello splicing alternativo di numerosi trascritti nei tessuti dei pazienti DM ed insorgenza del fenotipo patologico. Verrà presentata una rassegna delle alterazioni di splicing nelle DM, con particolare riferimento all’mRNA del gene BIN1, che gioca un ruolo chiave nella formazione delle invaginazioni tubulari del sarcolemma, alla base della biogenesi dei tubuli T (strutture membranose essenziali, nel tessuto muscolare striato, per il corretto accoppiamento eccitazione/contrazione). Le alterazioni nello splicing di BIN1 nei pazienti affetti da DM, dovuto ad una perdita di funzione della proteina MBNL1, hanno come conseguenza l’espressione di un a forma inattiva della proteina BIN1, priva di attività di legame per i fosfoinositidi e della capacità di formare invaginazioni tubulari della membrana plasmatica. Introducendo in un modello murino normale un simile difetto di splicing per BIN1 si ottengono alterazioni dei tubuli T e diminuita forza muscolare: ciò suggerisce che l’alterazione dello splicing per questo gene possa direttamente determinare l’insorgenza di debolezza muscolare, una delle caratteristiche più significative delle DM.

(*)

UPMC Univ Paris 6, UM 76, Institut de Myologie and Inserm, U974 and CNRS, UMR7215, 47/83 bld de l’hopital, F-75013, Paris, France. E-mail : denis.furling@upmc.fr


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*** ABSTRACT. – Myotonic dystrophy of type 1 (DM1) is one of the most common muscular dystrophy in adults characterized by progressive muscle wasting and weakness, myotonia, cardiac conduction defects, alteration in cognitive functions as well as several other multisystemic symptoms. DM1 is an autosomal dominant inherited disease caused by an unstable CTG expansion ranging from ~50 to more than 1,000 repeats in the 3’ non-coding region of the DMPK gene. Expression of DMPK RNAs with expanded CUG repeats supports a toxic RNA gain-of-function as a pathologic mechanism for DM1. A similar or common mechanism may also be involved in DM type 2 that is caused by CCTG expansion in the first intron of the CNP (ZNF9) gene and shares similar clinical features with DM1 disease. In both myotonic dystrophies, nuclear accumulation of pathogenic CUG/CCUGexp-RNAs alters the activities of the RNA binding proteins such as MBNL1 and CUG-BP1 that leads to alternative splicing mis-regulation of a numerous of transcripts in DM tissues and ultimately, to clinical features of the disease. An overview of the DM splicing mis-regulation will be presented, with focus on mis- regulation of the BIN1 mRNA. In muscle, BIN1 plays an important role in tubular invaginations of the plasma membrane and is required for biogenesis of T-tubules, which are specialized membrane structures essential for excitation-contraction coupling. BIN1 splicing mis-regulation in DM patients due to MBNL1 loss-of-function results in the expression of an inactive form of BIN1 deprived of phosphoinositide-binding and membrane-tubulating activities. Reproducing similar BIN1 mis-splicing defect in the muscles of wild type mice is sufficient to promote T-tubule alterations and muscle strength decrease, suggesting that alteration of BIN1 splicing may contributes to muscle weakness, a prominent feature in DM.

KEY WORDS. – myotonic dystrophy, CTG repeats, RNA, alternative splicing, weakness.

INTRODUCTION Myotonic dystrophy type 1 (DM1) also called Steinert disease (MIM#160900) is one of the most common muscular dystrophies encountered in adults. Progressive muscle wasting and weakness, myotonia, cardiac conduction defects, alteration in cognitive functions as well as several other multisystemic symptoms characterize this dominantly inherited disease (Harper 2001). The DM1 mutation was identified in 1992 and this complex disease is caused by an expanding (CTG)n repeat of 50 to several thousand triplets in the 3’non-coding region of the dystrophia myotonica-protein kinase (DMPK) gene on chromosome 19 (Brook et al. 1992; Mahadevan et al. 1992). Unaffected individuals have fewer than 38 repeats. The size of the expansion is generally correlated with the clinical severity and the age of onset of the


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disease (Hunter et al. 1992; Tsilfidis et al. 1992; Groh et al. 2011). Due to the variable clinical symptoms, several forms of the disease (asymptomatic late-onset, mild adult-onset, childhood-onset and congenital) have been described. The severe congenital form is associated with large expansions (over 1500 CTG repeats), and affected patients have motor and mental retardation. The disease-associated repeat expansion is very unstable and the number of triplets increases across generations providing a molecular basis for the anticipation phenomenon observed in DM1 families (Harper et al. 1992; Lavedan et al. 1993). In addition to intergenerational instability, CTG repeat expansion is also unstable in somatic tissues throughout the lifetime of the patient. Evidences for an RNA gain-of-function mechanism in DM1 pathogenesis came to light progressively. Both wild-type and mutant DMPK alleles are transcribed into mRNAs but mutant transcripts with expanded CUG repeats (CUGexp-RNAs) are sequestered in the nucleus as discrete aggregates or foci leading to decrease cytoplasmic DMPK mRNA levels (Taneja et al. 1995; Davis et al. 1997). Subsequent reduction of DMPK protein levels has been a subject of controversy but reduced DMPK levels were observed in muscles samples from DM1 patients as well as DM1 muscle cells (Fu et al. 1992; Maeda et al. 1995; Furling et al. 2001; Furling et al. 2003). Possible involvement of DMPK haploinsufficiency in DM1 pathophysiology as well as reduced levels of SIX5 observed in DM1 tissues due to the SIX homeobox 5 (SIX5) gene location directly downstream from the DM1 locus, were first investigated by generating mouse models. However heterozygous Dmpk or Six5 knockout mice failed to reproduce DM1-like symptoms suggesting that DMPK or SIX5 haploinsufficiency are probably not responsible for the DM1 phenotype (Jansen et al. 1996; Reddy et al. 1996; Klesert et al. 2000; Sarkar et al. 2000). Afterwards it has been suggested that the mutant transcripts from the expanded DMPK allele were pathogenic per se. Animal models were developed to investigate the role and the deleterious effects of CUGexp-RNA expression. Transgenic mice that expressed CUG repeat expansion either in the 3’UTR of the human skeletal muscle alpha actin (HSA-LR) mRNA (Mankodi et al. 2000) or in its natural context within the 3’UTR of the human DMPK transcript (Seznec et al. 2001), exhibited several DM1 features including nuclear aggregates of CUGexp-RNA, myotonia discharges and muscle abnormalities. In addition, severe muscle wasting was described in an inducible EpA960/HSA-Cre-ER transge-


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nic mice expressing 960 interrupted CTG repeats within the context of the DMPK exon 15 (Orengo et al. 2008) and progressive muscle atrophy was observed in mice expressing human DMPK mRNA with 550 CUG repeats (Vignaud et al. 2010). Altogether, these studies provided strong experimental supports for a key role of CUGexp-RNAs in DM1 pathogenesis. The last evidence for a RNA gain-of-function mechanism came from the identification of a myotonic dystrophy type 2 disorder (DM2; MIM#602668) that shares similar clinical features with DM1 disease suggesting a common molecular mechanism. DM2 is caused by a (CCTG)n repeat expansion ranging from 100 to 11.000 units in the first intron of the CCHC-type zinc finger, nucleic acid binding protein (CNBP also known as ZNF9) gene, a non-coding region from a gene non-related to DMPK (Liquori et al. 2001). The RNAs containing the expanded CCUG repeats are also retained in the nucleus and formed aggregates providing an additional support for a central role of mutant RNAs containing expanded repeats in pathophysiology of both DM1 and DM2 diseases. The CUGexp-RNAs are not exported into the cytoplasm but are retained in the nuclear compartment as discrete aggregates or foci that are easily detected by FISH (Taneja et al. 1995). The mutant DMPK mRNAs are spliced and polyadenylated but their nuclear sequestration due to expanded CUG repeats in the 3’UTR, prevents any translation (Davis et al. 1997). Within the nuclei, the foci of CUGexp-RNAs are localized at the periphery of the nuclear speckles, which are structures enriched in splicing snRNPs and the spliceosome assembly factor SC35 as well as many other transcription and splicing-related factors (Holt et al. 2007). The pathogenic DMPK transcripts do not enter into the speckles (Smith et al. 2007) suggesting that their export is blocked at an early step in nucleoplasmic transport. In vitro studies including crystal structure, enzymatic mapping, optical melting and electron microscopy, have demonstrated that expanded CUG repeats are able to form stable hairpin structures (Michalowski et al. 1999; Miller et al. 2000; Tian et al. 2000; Mooers et al. 2005). These double-stranded structures are defined by Watson-Crick G-C base-pairs separated by a periodic U-U mismatch. The muscleblind-like 1 (MBNL1) proteins were found to bind these expanded CUG repeats and to colocalize with nuclear foci of CUGexp-RNAs in DM1 cells (Miller et al. 2000). MBNL1 silencing by RNA interference significantly reduces the number of foci and restores the capacity of


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these pathogenic transcripts to progress through the nuclear speckles (Smith et al. 2007) indicating that the binding of MBNL1 to the abnormal CUG repeats may promote nuclear foci formation (Miller et al. 2000; Dansithong et al. 2005; Smith et al. 2007). It should be noted that MBNL1 also colocalizes with the nuclear foci of CCUG expanded RNA in DM2 cells however the DM2 foci are not localized at the periphery of the nuclear speckles as observed for the DM1 foci (Holt et al. 2007). Besides the difference within the expanded nucleotide repeat (CUG vs. CCUG) between DM1 and DM2, the entrapped RNAs in DM2 may contain intronic expanded CCUG repeats only since the CNBP pre-mRNA seems to be normally spliced (Margolis et al. 2006) and/or expanded CCUG repeats into abnormally spliced CNBP transcripts (Raheem et al. 2010). Finally, nuclear retention of the CUGexpRNAs participates to the pathogenic mechanism since the nuclear export of an artificial CUGexp-RNAs by inclusion of woodchuck posttranscriptional regulatory element reduces cellular defects (Mastroyiannopoulos et al. 2005) and the expression of DM1 foci exclusively in the cytoplasmic compartment does not induce key DM1 features in a mouse model (Dansithong et al. 2008). At the molecular level, one of the best-characterized transdominant effects induced by the CUGexp-RNAs in DM1 is the misregulation of alternative splicing of a subset of pre- mRNAs. To date, more than twenty-five transcripts have been found to be mis-spliced in different tissues of DM1 patients (Osborne and Thornton 2006). The misregulation of splicing events in DM1 is distinct from aberrant splicing caused by mutations in regulatory splicing sites that lead to the expression of aberrant mRNA. In DM1, mis-splicing events result from an inappropriate regulation of alternative splicing due to altered activities of splicing regulators such as MBNL1 and CELF1: - the MBNL1 RNA binding protein has been shown to bind, in a length-dependant manner, CUGexp-RNA with high affinity and form ribonucleoprotein complexes (Miller et al. 2000). MBNL1 is part of a conserved MBNL family including MBNL1, 2, 3, and all members contain four CCCH zinc-finger protein domains that are structured in pairs and acted as RNA binding domains (Pascual et al. 2006). Sequestration of MBNL1 within the nuclear aggregates of CUGexp-RNAs and the subsequent involvement of MBNL1 lossof-function in DM1 pathogenesis has been supported by the generation of a knockout Mbnl1 mouse model that demonstrates a DM-


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like phenotype as well as alternative splicing misregulation (Kanadia et al. 2003). Moreover a majority of the modifications in alterative splicing observed in the HSA-LR mice expressing CUGexp-RNA can be attributed to the loss-of-function of the MBNL1 splicing factor (Osborne et al. 2009; Du et al. 2010). In addition, mis-splicing events observed in this DM1 mouse model as well as myotonia can be reversed by MBNL1 overexpression in skeletal muscles (Kanadia et al. 2006). Several reports have demonstrated the regulatory splicing function of MBNL1 on several DM1 transcripts such as CLC1, cTNT or IR. Now, it is established that MBNL1 loss-of-function due to its sequestration by the CUGexp-RNA contributes to the “spliceopathy� in DM1. - CELF1 (also known as CUGBP1) is another RNA binding protein involved in this process. This factor is a member of the CELF family that contains 6 proteins with high homology (Barreau et al. 2006). Interestingly, CELF1 and MBNL1 are antagonistic regulators of many splicing events that are mis-regulated in DM1. CELF1 is able to bind single-strand CUG repeats but does not colocalize with the nuclear aggregates of CUGexp-RNA in DM1 cells and is not sequestered like MBNL1 (Timchenko et al. 2001). In contrast, the level of CELF1 is increased in DM1 tissues leading to a gain of CELF1 activity. It has been shown that the expression of CUGexp-RNA results in hyperphosphorylation and stabilization of the CELF1 protein through an inappropriate activation of the Protein Kinase C (Kuyumcu-Martinez et al. 2007; Wang et al. 2009). The pathogenic role of CELF1 in DM1 was supported by the fact that transgenic mice overexpressing CELF1 reproduce splicing misregulation as well as DM1 muscle features (Koshelev et al. 2010; Ward et al. 2010). Furthermore, increased levels of CELF1 is also found in the DM1 mouse model expressing inducible 960 interrupted CTG repeats, which exhibits muscle wasting as well as splicing defects that are only related to CELF1 (e.g. Capzb, Mfn2, Ank2 and Fxr1h) and not to MBNL1, suggesting that the elevation of CELF1 could participate to the DM1 muscle phenotype (Orengo et al. 2008). MBNL1 and CELF1 factors are developmental regulators of splicing events especially during the fetal to adult transition, and the modification of their activities in DM1 tissues leads to the expression of a fetal splicing pattern in adult tissues (Lin et al. 2006; Kalsotra et al. 2008). It should be noted that altered expression of splicing factors and


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alternative splicing changes may also occur during active regeneration process in degenerative muscle diseases (Orengo et al.). However no massive degeneration/regeneration was observed in DM1 muscles (Thornell et al. 2009) and altered splicing events were found in nonregenerating tissue such as DM1 cardiac tissue (Philips et al. 1998; Wang et al. 2011) confirming that misregulation of alternative splicing in DM1 is more likely a primary response to the expression of CUGexp-RNAs rather than a secondary effect to robust degeneration/regeneration process. The first splicing misregulation described in DM1 cardiac muscle was the abnormal inclusion of exon 5 in cTNT (Philips et al. 1998). Since then, several other transcripts with inappropriate splicing patterns have been identified in both skeletal muscle and brain (see Table 1) including those coding for the insulin receptor (IR) (Savkur et al. 2001), the muscle specific chloride channel (CLC1)(Charlet et al. 2002; Mankodi et al. 2002), the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 1 and 2, the ryanodine receptor (Kimura et al. 2005), the myotubularine-related protein 1(Buj-Bello et al. 2002), the tau protein (Sergeant et al. 2001), and the N-methyl-alpha-aspartate receptor (Jiang et al. 2004). Among the known mis-splicing events, most of them may participate to the pathologic process but very few have been directly correlated with disease symptoms. One of the exceptions is the CLC-1 splicing defect, which has been associated with myotonia, a characteristic feature of the disease. This splicing misregulation leads to the inclusion of exon 7a and subsequently to a truncated CLC1 protein that is devoid of channel activity and is not correctly addressed to the membrane of the muscle fibers, resulting in reduced muscle chloride conductance and myotonia (Charlet et al. 2002; Mankodi et al. 2002). Consistent with the RNA gain-of-function hypothesis that altered MBNL1 activity, both MBNL1 knockout mice and HSA-LR mice that express CUGexp-RNA showed Clc-1 splicing misregulation, loss of Clc-1 channel at the membrane, and myotonia. Finally, correction of this sole splicing defect by using antisense oligonucleotide that force the skipping of exon 7a in the muscle of HSA-LR mice abolished myotonia (Wheeler et al. 2007) confirming the key role of Clc-1 mis-splicing in the myotonic phenotype in DM1. More recently, the newly identified BIN1 splicing defect has been associated with muscle weakness, another hallmark of DM1 (Fugier et al. 2011). This splicing defect was identified in collaboration with N. Charlet by using a whole genome approach (Affymetrix exon array).


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Table 1. Tisues

Pre-mRNA

Exon/intron deregulation

Inclusion/ exclusion

Ref.

Skeletal

Insulin receptor (INSR)

Exon 11

Exclusion

Savkur et al., 2001

Muscle

Chloride channel (CLCN1)

Intron 2

Inclusion

Charlet et al., 2002 Mankodi et al., 2002

Exon 7A

Inclusion

Lueck et al., 2006

BIN1 (Amphyphisine 2)

Exon 11

Exclusion

Fugier et al., 2011

Calcium channel (Ca(V)1.1)

Exon 29

Exclusion

Tang et al., 2012

Skeletal Troponin T (TNNT3)

Exon fœtal

Inclusion

Kanadia et al., 2003

Ryanodine recptor (RyR1)

Exon 70

Exclusion

Kimura et al., 2005

Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase1 (SERCA1)

Exon 22

Exclusion

Sarcoplasmic/endoplasmic réticulum Ca2+ ATPase2 (SERCA2)

Intron 19

Inclusion

LIM domain inding 3 (LB, ZASP)

11

Inclusion

Titin (TTN)

Zr4

Inclusion

Zr5

Inclusion

Nebulin-related anchoring protein (NRAP)

12

Inclusion

Calpaïn 3 (CAPN3)

16

Exclusion

Attractin-like (ATRNL1, ALP)

5a et 5b

Inclusion

Forming homology 2 domain containing 1 (FHOD1)

11a

Exclusion

Glutamine-fructose-6-phosphate transaminase 1 (GFPT1)

10

Exclusion

MBNL1

7

Inclusion

MBNL2

7

Inclusion

SET and MYND domain containing 1 (SMYD1)

39

Inclusion

Sperm associated antigen 9

39

Inclusion

Myotubularin-related protein 1 (MTMR1)

Exon 2.1

Exclusion

Buj-Bello et al., 2002

Exon2.3

Exclusion

Ho et al., 2005

Nakamori et al., 2008

Alpha-dystrobrevin (DTNA) Brain

Lin et al., 2006

Tau (MAPT)

Exon 11a

Inclusion

Exon 12

Inclusion

Exon 2

Exclusion

Exon 3

Exclusion

Exon 6

Exclusion exon 6c

Du et al., 2010

Sergean et al., 2001 Leroy et al., 2006

Inclusion exon 6d

Récepteur-N-methyl-Daspartate (NMDAR1)

Exon 10

Exclusion

Sergeant et al., 2001 Jiang et al., 2004

Exon 5

Inclusion

Jiang et al., 2004

Amyloid precursor protein (APP)

Exon 7

Exclusion

MBNL1

Exon 5

Inclusion

Dhaenens et al., 2010


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New as well as previously described splicing alteration events were identified in congenital DM1 muscle cells containing large CTG expansion and confirmed in skeletal muscles from DM patients. We focus our attention on BIN1 exon 11 splicing misregulation since mutation in this gene leads to Centronuclear Myopathy that share some similar features with the severe congenital form of DM1. BIN1 is a protein specialized in membrane curvature, whose function is regulated by alternative splicing. In skeletal muscles, inclusion of the muscle-specific exon 11, which encodes a phosphoinositide-binding (PI) domain, generates an isoform of BIN1 that induces tubular invaginations of membranes and is implicated in T- tubules biogenesis. The muscle T-tubule network is a specialized membrane structure fundamental for excitation-contraction (E-C) coupling, and the disruption of BIN1 in Drosophila leads to severely disorganized T-tubules and defects of the E-C coupling machinery. We demonstrate that MBNL1 binds to BIN1 pre-mRNA and regulates its alternative splicing. BIN1 splicing misregulation results in expression of an inactive form of BIN1 deprived of PtdIns5P-binding and membrane-tubulating activities. Consistent with a defect of BIN1, muscle T-tubules are altered in DM patients and membrane structures are restored upon expression of the normal splicing form of BIN1 in DM1 muscle cells. In non- affected muscles, BIN1 is organized in transversal projections and co-localized with the L- type calcium channel CACNA1S. In DM1 muscles, BIN1 was disorganized and presented a more diffuse localization. Ultrastructural analysis confirmed alterations of the T-tubule network with presence of irregular and longitudinally orientated T-tubules. To test the contribution of BIN1 splicing alteration for DM phenotype, we artificially forced exon 11 skipping in mouse skeletal muscle using an U7-snRNA exon-skipping strategy. Artificial skipping of Bin1 exon 11 promotes Bin1 mis-localization but no major atrophy or degeneration of muscle fibers. However ~30% of T-tubules were abnormal in Bin1 exon 11 skipped muscles, with longitudinally orientated, disorganized and irregular structures suggesting that alteration of the T-tubule network. No significant muscle mass loss was observed but isometric strength measurement showed that skipping of Bin1 exon 11 induced a ~28% decreased of specific muscle strength. Our results suggest that splicing mis-regulation of BIN1 and of other pre-mRNAs involved in E-C coupling ultimately results in muscle weakness in DM patients. Interestingly, a recent report (Tang et al. 2012) proposed that mis-spli-


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DENIS FURLING

cing of the Cac1.1 that altered the function of this calcium channel is also associated with muscle weakness and may exacerbate DM1 myopathy. Altogether, these data suggest that a common mechanism, involving BIN1 and alteration of the calcium homeostasis coupled to the excitation-contraction process, may underlie muscle weakness in DM1. Over the years, the RNA gain-of-function hypothesis has progressively emerged as a pathogenic mechanism for the complex DM1 disease. Alternative splicing misregulation of several pre-mRNAs due to the altered activities of MBNL1 and CELF1 RNA binding proteins by CUGexp-RNAs, contributes to the DM1 pathophysiology. However it seems unlike that it can explain the wide spectrum of DM1 clinical symptoms. The CUGexp-RNAs have effects in trans and may alter other processes at both transcriptional and post-transcriptional levels. Indeed, altered activities of the MBNL1 and CELF1 may affect other RNA-processing events regulated by these RNA binding proteins. Thus, the activity of CELF1 varies depending on its cellular localization. In the nucleus, CELF1 acts as a splicing regulator whereas in the cytoplasm, it can regulate the translational activity of proteins like p21 and MEF2A, which are involved in muscle cell differentiation (Iakova et al. 2004; Timchenko et al. 2004). A concomitant translational deregulation of CELF1 targets and associated functions indicate that other post-transcriptional mechanisms could also be altered by the CUGexpRNAs. Unlike CELF1, no effect on translation has been described for MBNL1 yet, even though MBNL1 is also present in the cytoplasmic compartment. Alternative splicing results in the production of several isoforms of MBNL1 and the associated protein isoforms have been shown to have either a nuclear or a nucleo-cytoplasmic localization (Tran et al. 2011). It should be noted that the splicing of MBNL1 itself is altered in DM1 leading to increased levels of exclusively the nuclear isoforms. However the impact of such alterations on the activities of MBNL1 is still not clearly understood. More recently, a novel function of MBNL1 as a regulator of the micro-RNA miR-1 biogenesis has been described (Rau et al. 2011). Consistent with MBNL1 sequestration and loss-of-function, miR-1 processing was altered in the hearts of DM1 patients as well as miR-1 targets such as CACNA1 and GJA1 that encode calcium and gap junction channels, respectively. Interestingly, other


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micro-RNAs were also deregulated in muscles of DM1 patients suggesting that the deregulation of this species of small non-coding RNA could have an impact on DM1 pathology (Gambardella et al. 2010; Perbellini et al. 2011). Finally, there is increasing evidence (Yadava et al. 2008; Osborne et al. 2009; Du et al. 2010; Marteyn et al. 2011) to suggest that the CUGexp-RNAs may also interfere with gene expression but further studies are required to determine the mechanism involved in this process and the pathophysiological consequences. By interfering with RNA metabolism of either coding or non-coding RNAs, the CUGexp-RNAs may act on the expression of various proteins in a tissue-specific manner and participate to the complex and multisystemic DM1 phenotype.

ACKNOWLEDGMENTS The author would like to thank the Association Franรงaise contre les Myopathies (AFM), UPMC, Inserm and CNRS.


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Holt I., S. Mittal, et al. (2007). Defective mRNA in myotonic dystrophy accumulates at the periphery of nuclear splicing speckles. Genes Cells 12(9): 1035-48. Hunter A., C. Tsilfidis, et al. (1992). The correlation of age of onset with CTG trinucleotide repeat amplification in myotonic dystrophy. J Med Genet 29(11): 774-9. Iakova P., G.L. Wang, et al. (2004). Competition of CUGBP1 and calreticulin for the regulation of p21 translation determines cell fate. Embo J 23(2): 406-17. Jansen G., P.J. Groenen, et al. (1996). Abnormal myotonic dystrophy protein kinase levels produce only mild myopathy in mice. Nat Genet 13(3): 316-24. Jiang H., A. Mankodi, et al. (2004). Myotonic dystrophy type 1 is associated with nuclear foci of mutant RNA, sequestration of muscleblind proteins and deregulated alternative splicing in neurons. Hum Mol Genet 13(24): 3079-88. Kalsotra A., X. Xiao, et al. (2008). A postnatal switch of CELF and MBNL proteins reprograms alternative splicing in the developing heart. Proc Natl Acad Sci U S A 105(51): 20333-8. Kanadia R.N., K.A. Johnstone, et al. (2003). A muscleblind knockout model for myotonic dystrophy. Science 302(5652): 1978-80. Kanadia R.N., J. Shin, et al. (2006). Reversal of RNA missplicing and myotonia after muscleblind overexpression in a mouse poly(CUG) model for myotonic dystrophy. Proc Natl Acad Sci U S A 103(31): 11748-53. Kimur T., M. Nakamori, et al. (2005). Altered mRNA splicing of the skeletal muscle ryanodine receptor and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase in myotonic dystrophy type 1. Hum Mol Genet 14(15): 2189-200. Klesert T.R., D.H. Cho, et al. (2000). Mice deficient in Six5 develop cataracts: implications for myotonic dystrophy. Nat Genet 25(1): 105-9. Koshelev M., S. Sarma, et al. (2010). Heart-specific overexpression of CUGBP1 reproduces functional and molecular abnormalities of myotonic dystrophy type 1. Hum Mol Genet 19(6): 1066-75. Kuyumcu-Martinez N.M., G.S. Wang, et al. (2007). Increased steady-state levels of CUGBP1 in myotonic dystrophy 1 are due to PKC-mediated hyperphosphorylation. Mol Cell 28(1): 68-78. Lavedan C., H. Hofmann-Radvanyi, et al. (1993). Myotonic dystrophy: size- and sexdependent dynamics of CTG meiotic instability, and somatic mosaicism. Am J Hum Genet 52(5): 875-83. Lin X., J.W. Miller, et al. (2006). Failure of MBNL1-dependent post-natal splicing transitions in myotonic dystrophy. Hum Mol Genet 15(13): 2087-97. Liquori C.L., K. Ricker, et al. (2001). Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9. Science 293(5531): 864-7. Maeda M., C.S. Taft, et al. (1995). Identification, tissue-specific expression, and subcellular localization of the 80- and 71-kDa forms of myotonic dystrophy kinase protein. J Biol Chem 270(35): 20246-9. Mahadevan M., C. Tsilfidis, et al. (1992). Myotonic dystrophy mutation: an unstable CTG repeat in the 3’ untranslated region of the gene. Science 255(5049): 1253-5. Mankodi A., E. Logigian, et al. (2000). Myotonic dystrophy in transgenic mice expressing an expanded CUG repeat. Science 289(5485): 1769-73.


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Mankodi A., M.P. Takahashi, et al. (2002). Expanded CUG repeats trigger aberrant splicing of ClC-1 chloride channel pre-mRNA and hyperexcitability of skeletal muscle in myotonic dystrophy. Mol Cell 10(1): 35-44. Margolis J.M., B.G. Schoser, et al. (2006). DM2 intronic expansions: evidence for CCUG accumulation without flanking sequence or effects on ZNF9 mRNA processing or protein expression. Hum Mol Genet 15(11): 1808-15. Marteyn A., Y. Maury, et al. (2011). Mutant human embryonic stem cells reveal neurite and synapse formation defects in type 1 myotonic dystrophy. Cell Stem Cell 8(4): 434-44. Mastroyiannopoulos N.P., M.L. Feldman, et al. (2005). Woodchuck post-transcriptional element induces nuclear export of myotonic dystrophy 3’ untranslated region transcripts. EMBO Rep 6(5): 458-63. Michalowski S., J.W. Miller, et al. (1999). Visualization of double-stranded RNAs from the myotonic dystrophy protein kinase gene and interactions with CUG-binding protein. Nucleic Acids Res 27(17): 3534-42. Miller J.W., C.R. Urbinati, et al. (2000). Recruitment of human muscleblind proteins to (CUG)(n) expansions associated with myotonic dystrophy. Embo J 19(17): 4439-48. Mooers B.H., J.S. Logue, et al. (2005). The structural basis of myotonic dystrophy from the crystal structure of CUG repeats. Proc Natl Acad Sci U S A 102(46): 16626-31. Orengo J.P., P. Chambon, et al. (2008). Expanded CTG repeats within the DMPK 3’ UTR causes severe skeletal muscle wasting in an inducible mouse model for myotonic dystrophy. Proc Natl Acad Sci U S A 105(7): 2646-51. Orengo J.P., A.J. Ward, et al. (2011). Alternative splicing dysregulation secondary to skeletal muscle regeneration. Ann Neurol 69(4): 681-90. Osborne R.J., X. Lin, et al. (2009). Transcriptional and post-transcriptional impact of toxic RNA in myotonic dystrophy. Hum Mol Genet 18(8): 1471-81. Osborne R.J. and C.A. Thornton (2006). RNA-dominant diseases. Hum Mol Genet 15 Suppl 2: R162-9. Pascual M., M. Vicente, et al. (2006). The Muscleblind family of proteins: an emerging class of regulators of developmentally programmed alternative splicing. Differentiation 74(2-3): 65-80. Perbellini R., S. Greco, et al. (2011). Dysregulation and cellular mislocalization of specific miRNAs in myotonic dystrophy type 1. Neuromuscul Disord 21(2): 81-8. Philips A.V., L.T. Timchenko, et al. (1998). Disruption of splicing regulated by a CUGbinding protein in myotonic dystrophy. Science 280(5364): 737-41. Raheem O., S.E. Olufemi, et al. (2010). Mutant (CCTG)n expansion causes abnormal expression of zinc finger protein 9 (ZNF9) in myotonic dystrophy type 2. Am J Pathol 177(6): 3025-36. Rau F., F. Freyermuth, et al. (2011). Antagonistic role of MBNL1 and LIN28 promotes specific alteration of pre-miR-1 processing and is associated with heart defects in Myotonic Dystrophic Patients. Nature structural and molecular biology in press. Reddy S., D.B. Smith, et al. (1996). Mice lacking the myotonic dystrophy protein kinase develop a late onset progressive myopathy. Nat Genet 13(3): 325-35. Sarkar P.S., B. Appukuttan, et al. (2000). Heterozygous loss of Six5 in mice is sufficient to cause ocular cataracts. Nat Genet 25(1): 110-4.


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Savkur R.S., A.V. Philips, et al. (2001). Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy.� Nat Genet 29(1): 40-7. Sergeant N., B. Sablonniere, et al. (2001). Dysregulation of human brain microtubuleassociated tau mRNA maturation in myotonic dystrophy type 1. Hum Mol Genet 10(19): 2143-55. Seznec H., O. Agbulut, et al. (2001). Mice transgenic for the human myotonic dystrophy region with expanded CTG repeats display muscular and brain abnormalities. Hum Mol Genet 10(23): 2717-26. Smith K.P., M. Byron, et al. (2007). Defining early steps in mRNA transport: mutant mRNA in myotonic dystrophy type I is blocked at entry into SC-35 domains. J Cell Biol 178(6): 951-64. Taneja K.L., M. McCurrach, et al. (1995). Foci of trinucleotide repeat transcripts in nuclei of myotonic dystrophy cells and tissues. J Cell Biol 128(6): 995-1002. Tang Z.Z., V. Yarotskyy, et al. (2012). Muscle weakness in myotonic dystrophy associated with misregulated splicing and altered gating of Ca(V)1.1 calcium channel. Hum Mol Genet 21(6): 1312-24. Thornell L.E., M. Lindstom, et al. (2009). Satellite cell dysfunction contributes to the progressive muscle atrophy in myotonic dystrophy type 1. Neuropathol Appl Neurobiol 35(6): 603-13. Tian B., R.J. White, et al. (2000). Expanded CUG repeat RNAs form hairpins that activate the double-stranded RNA-dependent protein kinase PKR. Rna 6(1): 79-87. Timchenko N.A., Z.J. Cai, et al. (2001). RNA CUG repeats sequester CUGBP1 and alter protein levels and activity of CUGBP1. J Biol Chem 276(11): 7820-6. Timchenko N.A., R. Patel, et al. (2004). Overexpression of CUG triplet repeat binding protein, CUGBP1, in mice inhibits myogenesis. J Biol Chem 279: 13129-39. Tran H., N. Gourrier, et al. (2011). Analysis of exonic-regions involved in nuclear localization, splicing activity and dimerization of muscleblind-like-1 isoforms. J Biol Chem. Tsilfidis C., A.E. MacKenzie, et al. (1992). Correlation between CTG trinucleotide repeat length and frequency of severe congenital myotonic dystrophy. Nat Genet 1(3): 192-5. Vignaud A., A. Ferry, et al. (2010). Progressive skeletal muscle weakness in transgenic mice expressing CTG expansions is associated with the activation of the ubiquitinproteasome pathway. Neuromuscul Disord 20(5): 319-25. Wang G.S., M.N. Kuyumcu-Martinez, et al. (2009). PKC inhibition ameliorates the cardiac phenotype in a mouse model of myotonic dystrophy type 1. J Clin Invest 119(12): 3797-806. Wang L.C., K.Y. Chen, et al. (2011). Muscleblind participates in RNA toxicity of expanded CAG and CUG repeats in Caenorhabditis elegans. Cell Mol Life Sci 68(7): 1255-67. Ward A J., M. Rimer, et al. (2010). CUGBP1 overexpression in mouse skeletal muscle reproduces features of myotonic dystrophy type 1. Hum Mol Genet 19(18): 3614-22. Wheeler T.M., J.D. Lueck, et al. (2007). Correction of ClC-1 splicing eliminates chloride channelopathy and myotonia in mouse models of myotonic dystrophy. J Clin Invest 117(12): 3952-7. Yadava R.S., C.D. Frenzel-McCardell, et al. (2008). RNA toxicity in myotonic muscular dystrophy induces NKX2-5 expression. Nat Genet 40(1): 61-8.


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CELL NUCLEAR ALTERATIONS IN MYOTONIC DYSTROPHY MANUELA MALATESTA (*), MARZIA GIAGNACOVO (**)

RIASSUNTO. – Nel nucleo cellulare i trascritti primari dei geni subiscono varie modificazioni molecolari per diventare RNA maturi, pronti per essere esportati nel citoplasma. Questi eventi molecolari sono ordinati cronologicamente e spazialmente, e avvengono prevalentemente in strutture contenenti ribonucleoproteine (RNP). Difetti nella maturazione dell’RNA sono stati associati a patologie che provocano distrofia muscolare: nelle distrofie miotoniche di tipo 1 (DM1) e di tipo 2 (DM2), le caratteristiche multisistemiche (ad esempio, miotonia, distrofia muscolare, cardiomiopatia dilatativa, difetti della conduzione cardiaca, cataratta, insulino-resistenza, anomalie sierologiche specifiche) che contraddistinguono queste patologie sono causate dall’espansione di due distinte sequenze nucleotidiche ((CTG)n nel gene DMPK del cromosoma 19q13 nella DM1, e (CCTG)n nel gene ZNF9 del cromosoma 3q21 nella DM2). Associando tecniche biomolecolari e citochimiche, è stato dimostrato che i meccanismi di base in entrambe le DM consistono nel sequestro nucleare degli RNA espansi: i trascritti contenenti CUG e CCUG si accumulano in foci intranucleari rispettivamente nella DM1 e nella DM2, ed alterano la regolazione e la localizzazione intranucleare delle proteine CUGBP1 e MBLN1, necessarie per la maturazione fisiologica del pre-RNA messaggero (mRNA). Mediante tecniche immunocitochimiche in microscopia ottica ed elettronica, il nostro gruppo ha dimostrato che, nella DM2, i foci contenenti MBNL1 sequestrano anche snRNPs e hnRNPs, fattori di splicing coinvolti nelle fasi precoci della maturazione dei trascritti, a supporto dell’ipotesi che il fenotipo patologico multifattoriale dei pazienti distrofici sia dovuto ad una generale alterazione dei processi maturativi posttrascrizionali del pre-mRNA. Abbiamo anche dimostrato che, nei muscoli scheletrici di pazienti affetti da DM1 e DM2, diversi fattori di splicing e di cleavage si accumulano

(*) Dipartimento di Scienze Neurologiche e del Movimento, Sezione di Anatomia e Istologia, Università degli Studi di Verona, Strada Le Grazie 8, 37134 Verona, Italy. Tel.: +39.045.8027157 - Fax: +39.045.8027163. E-mail: manuela.malatesta@univr.it (**) Dipartimento di Biologia e Biotecnologie “Lazzaro Spallanzani”, Laboratorio di Biologia cellulare e Neurobiologia, Università degli Studi di Pavia, Pavia, Italy.


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nei mionuclei, suggerendo disfunzioni nei processi di maturazione del pre-mRNA simili alle alterazioni nucleari tipiche della sarcopenia (la perdita di massa e funzione muscolare che si verifica fisiologicamente con l’invecchiamento). Inoltre, in uno studio in vitro, abbiamo osservato che i mioblasti derivati da cellule satellite di pazienti affetti da DM2 mostrano caratteristiche tipiche della senescenza e disfunzioni dei processi maturativi del pre-mRNA più precocemente dei mioblasti di individui sani. Nel loro insieme, questi dati suggeriscono l’esistenza di meccanismi nucleari comuni alla base delle alterazioni muscolari in diverse condizioni patologiche. *** ABSTRACT. – In the cell nucleus, genes are transcribed, and the primary transcripts undergo molecular processing which generates mature RNAs to be exported to the cytoplasm. The events leading to the formation of mature RNAs are chronologically and spatially ordered, and they mostly occur on distinct ribonucleoprotein (RNP)-containing structures. Defects in the RNA maturation pathways have been related to diseases leading to muscle dystrophy: in myotonic dystrophy type 1 (DM1) and type 2 (DM2), the characteristic multisystemic features (e.g., myotonia, muscular dystrophy, dilated cardiomyopathy, cardiac conduction defects, cataracts, insulin-resistance, and disease-specific serological abnormalities) are caused by the expansion of two distinct nucleotide sequences: (CTG)n in the 3’ untranslated region of the DMPK gene on chromosome 19q13 in DM1, and (CCTG)n in the first intron of the ZNF9 gene on chromosome 3q21 in DM2. Combining biomolecular and cytochemical techniques, it has been demonstrated that the basic mechanisms of both DMs reside in the nuclear sequestration of the expanded RNAs: CUG- and CCUG-containing transcripts accumulate in intranuclear foci in DM1 and DM2 cells respectively, and alter the regulation and intranuclear localization of the RNA-binding proteins CUGBP1 and MBLN, which are necessary for the physiological processing of pre-mRNA. Using immunocytochemical techniques at light and electron microscopy, we have demonstrated that MBNL1-containing foci in DM2 cells also sequester snRNPs and hnRNPs, splicing factors involved in the early phases of transcript processing; this strengthens the hypothesis that the multifactorial phenotype of dystrophic patients could be due to a general alteration of the pre-mRNA post-transcriptional pathway. Interestingly, we also demonstrated that, in skeletal muscles of DM1 and DM2 patients, splicing and cleavage factors accumulate in myonuclei, suggesting an impairment of pre-mRNA processing reminiscent of the nuclear alterations typical of sarcopenia (i.e. the loss of muscle mass and function physiologically occurring during ageing). Moreover, in an in vitro study, we observed that satellite-cell-derived DM2 myoblasts show cell senescence alterations and impairment of the pre-mRNA maturation pathways earlier than the myoblasts from healthy patient. These results suggest possible common cellular mechanisms responsible for skeletal muscle wasting in different pathologies.


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CELL NUCLEAR ARCHITECTURE AND IN SITU ANALYSIS OF RNA PROCESSING In eukaryotic cells, gene primary transcripts (pre-mRNAs) undergo extensive modifications before generating mature mRNA to be exported to the cytoplasm. This processing occurs in the spliceosome, a molecular complex composed by five small nuclear ribonucleoproteins (snRNPs) (U1, U2, U4/U6 and U5 snRNPs) and many nonsnRNP splicing factors as well as by a large number of regulating factors. After removal of introns, the maturation of 3’ ends must be completed, and this requires the involvement of several cleavage and polyadenylation factors (Wahle and Rüegsegger, 1999). The events leading to the formation of mature mRNA occur mostly co-transcriptionally, thereby implying the simultaneous presence of different molecules on the transcriptional sites. The structural counterpart of these molecular complexes is represented by the perichromatin fibrils (PF), fine fibrillar structures mainly distributed at the edge of condensed chromatin (the so-called perichromatin region); PF represent the in situ form of nascent transcripts (reviews in Fakan, 1994, 2004) as well as of their splicing (Cmarko et al., 1999) and 3’ end processing (Schul et al., 1996; Cardinale et al., 2007). PF are therefore highly sensitive markers not only for monitoring the transcriptional and processing rate of a cell, but also for identifying the maturation level of (pre)mRNA occurring in the nucleus in functional correlation with the cellular metabolic state (Biggiogera et al., 2008). The pathways followed by the maturating pre-mRNA before to be exported to the cytoplasm are not completely known: it is likely that one part would migrate through the interchromatin space towards the nuclear pores as PF, while another part would be carried by the perichromatin granules (PG). These roundish RNP structures, preferentially occurring in the perichromatin region (Monneron and Bernhard, 1969), would act as both vectors and storage sites of already spliced pre-mRNA (Fakan, 1994, 2004), and modifications in the number and/or molecular composition of PG are markers of alterations of the intranuclear pathways (e.g. Puvion et al., 1977; Puvion-Dutilleul and Puvion, 1981; Lafarga et al., 1993; Zancanaro et al., 1993). The interchromatin granules (IG), occurring as clusters in the interchromatin space, represent storage, assembly and phosphorylation sites for


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transcription and splicing factors, but they are not directly involved in pre-mRNA modifications (Puvion and Puvion-Dutilleul, 1996; Bogolyubov et al., 2009). Therefore, the events leading to the formation of mature RNAs are chronologically and spatially ordered, and they mostly occur on distinct RNP-containing structures (Figure 1). The specific intranuclear location of these RNP components, clearly recognizable by the cytochemical EDTA technique (Bernhard, 1969), is a necessary prerequisite for the correct processing of nuclear RNAs to occur, so that whenever transcription and/or splicing are altered, the organization, composition, and intranuclear location of RNP-containing structures are also affected (Biggiogera et al., 2004, 2008; Malatesta et al., 2008, 2010a). As a consequence, the in situ analysis of the nuclear organization and molecular composition not only provides information about the DNA/RNA pathways which govern the cellular metabolism, but may reveal the occurrence of dysfunctions related to pathological phenotypes, also in skeletal muscle diseases (Malatesta and Meola, 2010).

CELL NUCLEAR ALTERATIONS IN MYOTONIC DYSTROPHY Myotonic dystrophies (DMs) are autosomal dominant disorders characterised by a variety of multisystemic features including muscular dystrophy with increased number of centrally located or clumped nuclei in muscle fibres (Vihola et al., 2003), myotonia (muscle hyperexcitability), dilated cardiomyopathy, cardiac conduction defects (Bachinski et al., 2003), insulin-resistance, cataracts (Meola and Moxley, 2004), and disease-specific serological abnormalities (Savkur et al., 2001, 2004; Day et al., 2003). Two forms of DM are known: the more severe DM1-Steinert’s disease (OMIM 160900) is caused by an expanded (CTG)n nucleotide sequence in the 3’ untranslated region of the Dystrophia Myotonic Protein Kinase (DMPK) gene (OMIM 605377) on chromosome 19q13 (Brook et al., 1992; Fu et al., 1992; Mahadevan et al., 1992); the second form, DM2 (OMIM 602688) displays a milder clinical phenotype and is caused by the expansion of the tetranucleotidic repeat (CCTG)n in the first intron of the Zinc Finger Protein (ZNF)-9 gene (OMIM 116955) (Liquori et al. 2001) on chromosome 3q21 (Ranum et al., 1998).


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Figure 1. An ultrathin section of a rat hepatocyte nucleus after glutaraldehyde fixation and Epon embedding. Following the EDTA staining which preferentially contrast RNP constituents (Bernhard, 1969), nucleoplasmic structures such as perichromatin fibrils (some are indicated by small arrows), perichromatin granules (large arrows) and clusters of interchromatin granules (open arrow) are easily visualized; also the nucleoli (Nu) remain well contrasted, whereas chromatin (Ch) is bleached and appears light grey. Bar: 1 Âľm. (From Fakan S, 2004. Eur J Histochem 48:5-14; reproduction authorized by the Editor)

Defects in the RNA maturation pathways have been related to both DM forms. Combining biomolecular and cytochemical techniques, it has been demonstrated that the expanded-CUG- and CCUG-containing transcripts in DM1 and DM2 cells are retained in the cell nucleus, and accumulate in the form of focal aggregates (Liquori et al., 2001). These


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nuclear foci of mutant mRNA specifically sequester the alternative splicing regulators CUG-binding protein 1 (CUGBP1) and muscleblind-like 1 (MBLN1) protein (Miller et al., 2000; Ladd et al., 2001; Fardaei et al., 2002; Holt et al., 2009; Jones et al., 2011), which are necessary for the physiological processing of pre-mRNA, especially for contractile protein synthesis (Llorian and Smith, 2011). The sequestration into foci leads to nuclear depletion and loss of function of these important factors (Mankodi et al., 2001). These focal aggregates are considered as a biomolecular feature of DMs, and have been detected in several adult tissues and cell cultures by in situ hybridization (Taneja et al., 1995; Liquori et al., 2001; Mankodi et al., 2003, 2005; Cardani et al., 2004, 2006, 2009; Schoser et al., 2004; Wheeler and Thornton, 2007). The molecular composition of nuclear foci is far from being fully elucidated. Our group, by combining immunofluorescence and immunoelectron microscopy on cultured myoblasts from DM patients, has demonstrated that foci also sequester hnRNPs and snRNPs, i.e. splicing factors involved in the early phases of the pre-mRNA processing (Perdoni et al., 2009) (Figures 2 and 3). The sequestration of these splicing factors strengthens the hypothesis that the multifactorial phenotype of dystrophic patients could be due to a general alteration of the pre-mRNA post-transcriptional pathway.

Figure 2. The immunolabelling for MBNL1 (red fluorescence in a) and that for the hnRNP core protein (green fluorescence in b) frequently co-locate (yellow fluorescence in the merged image, c). In the insets in panel c), the co-labelled foci indicated by the arrows are shown at a higher magnification. In panel a), the counterstaining with Hoechst 33258 of nuclear DNA is also shown (blue fluorescence). Bar: 10 Âľm.

In a very recent study, by using biochemical, immunocytochemical and morphometric techniques, we investigated the fate of MBNL1-containing foci in proliferating and in non-cycling cultured skin fibroblasts


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from DM2 patients (Giagnacovo et al., 2012). We demonstrated that MBNL1 associates to the transcribed RNA expanded sequences and accumulate in the nuclear foci during interphase; at mitosis, the foci relocate to the cytoplasm where they remain until the following early G1, when cytoplasmic foci undergo degradation; at the same time, newlyformed foci develop in the nucleus as a consequence of de novo accumulation of expanded RNAs. In DM2 proliferating fibroblasts, the cyclic release from the nucleus of the foci and their cytoplasmic degradation actually prevent a massive intranuclear sequestration of MBNL1 and other protein factors involved in pre-mRNA processing. On the contrary, when fibroblasts loose their proliferation capabilities and stop cycling, the nuclear foci do not undergo relocation/degradation and undergo progressive increase in number and size, due to a continuous accumulation of both expanded RNAs and protein factors among which MBNL1.

Figure 3. Transmission electron micrographs of myoblast nuclei from DM2 patients. a): Conventional ultrastructural morphology (glutaralaldehyde-fixation, OsO4 post-fixation, Epon embedding), lead citrate staining: the focus appears as a strongly electron-dense roundish nuclear domain (arrow) located in the nucleoplasm. b): Immunoelectron microscopy (paraformaldehyde fixation, Unicryl-embedding), EDTA staining: dual immunolabelling with anti-MBNL1 (12 nm gold particles) and anti-snRNP (6 nm gold particles) antibodies; the probes co-locate in the focus (arrow) and on perichromatin fibrils (arrowhead). Nu, nucleolus. Bars: 0.5 Âľm.

Interestingly, measurements on muscle biopsies taken from the same DM2 patients at different ages demonstrated that, in the nuclei of myofibres, the MBNL1-containing foci become larger with increasing patient’s age (Giagnacovo et al., 2012). This dynamic behaviour of nuclear foci is compatible with the evidence that in DM patients the most affected organs or tissues are those


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where non-renewing cells are mainly present, such as the skeletal muscle, heart and the central nervous system, whereas cells from self-renewing tissues (such as skin fibroblasts or layering epithelial cells) are much less affected. In addition to the formation of intranuclear foci, DM1 and DM2 cells show an altered distribution of nuclear RNP-containing structures and molecular factors responsible for pre-mRNA transcription and maturation. In particular, by means of ultrastructural immunocytochemistry on skeletal muscle biopsies from DM1 and DM2 patients, we have recently demonstrated (Malatesta et al., 2011b) that splicing and cleavage factors accumulate in the intranuclear functional sites where they are usually located (i.e., hnRNPs and CStF on PF; snRNPs on PF and IG) (Figure 4). Accordingly, new results on the intranuclear distribution of MBNL1 in myonuclei of skeletal muscle from DM1 and DM2 patients revealed an accumulation of this alternative splicing factor on PF and also on IG (where it does not usually occur in healthy subjects) (Malatesta et al., manuscript in preparation). This accumulation could hamper the functionality of the splicing machinery and slow down the intranuclear molecular trafficking thus reducing the metabolic activity of myonuclei, consistent with recent findings demonstrating a reduced protein synthesis in DM1 and DM2 myoblasts (Salisbury et al., 2009; Huichalaf et al., 2010).

Figure 4. Myonuclei of biceps brachii biopsies from healthy (a), DM1 (b) and DM2 (c) patients; double immunolabelling with anti-(Sm)snRNP (6 nm gold particles) and antihnRNP (12 nm gold particles) antibodies; EDTA staining. Both probes label perichromatin fibrils (arrows); in addition, the anti-(Sm)snRNP antibody labels interchromatin granules (IG). Note the lower labelling density in the healthy sample (a) in comparison to DM1 (b) and DM2 (c) myonuclei. Ch, condensed chromatin; arrowheads, perichromatin granules. Bars: 0.5 Âľm.


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MYOTONIC DYSTROPHY AND AGEING: COMMON MECHANISMS FOR SIMILAR EFFECTS?

Ageing is associated with a progressive loss of muscle mass, strength and function, known as sarcopenia (Ryall et al., 2008; Thompson, 2009; Cruz-Jentoft et al., 2011). Interestingly, under this condition the skeletal muscle is characterized by grouped atrophy, fiber size variability and centrally located nuclei (Endstrom et al., 2007), similarly to muscles affected by DM. In addition, some cell nuclear alterations described in myofibres of sarcopenic muscles are reminiscent of those found in DM cells. In fact, although no foci have been observed, factors involved in pre-mRNA post-transcriptional processing have been found to accumulate on PF and, sometimes, to move to IG, where they do not usually locate (Malatesta et al., 2009, 2010b, 2011a). This intranuclear accumulation/delocalization of RNP structures containing splicing and cleavage factors has been found not only in aged skeletal muscle but also in other tissues (e.g., liver, brain) from old rodents (Malatesta et al., 2003, 2004, 2005, 2007, 2010a; BertoniFreddari et al., 2004). This suggests that in ageing cells the entire production chain of mRNA, from synthesis to cytoplasmic export, becomes less efficient, likely contributing to the reduced cell responsiveness to metabolic stimuli which is typical of elderly. This loss of responsiveness would be particularly serious in skeletal muscle cells, where a misregulated protein turnover would result in a structural imbalance between muscle protein synthesis and degradation (review in Koopman, 2009). In a recent study (Malatesta et al., 2011c) we investigated in vitro the structural and functional features of satellite-cell-derived myoblasts and we observed that DM2 myoblasts show cell-senescence alterations such as cytoplasmic vacuolisation, reduction of the proteosynthetic apparatus, accumulation of heterochromatin and impairment of the pre-mRNA maturation pathways earlier than myoblasts from healthy patients (Figures 5 and 6); moreover, DM2 myoblasts generate myotubes characterised by structural defects similar to senescent healthy myotubes (Giagnacovo et al., 2011). The early occurrence of senescence-related features in satellite cellderived myoblasts would therefore reduce the regeneration capability of DM2 satellite cells, thus contributing to the muscular dystrophy in DM2 patients.


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Figure 5. Phase contrast and fluorescence micrographs of myoblasts from healthy (a,a’ and b,b’) and DM2 patients (c,c’ and d,d’) at the second (a and c) and the fourteenth (b and d) passage in culture, after immunolabeling for desmin. Myoblasts are characterized by their thin and elongated shape, and exhibit immunopositivity for desmin (green fluorescence). Nuclear DNA was counterstained with Hoechst 33258 (blue fluorescence). It is worth noting that already at an early passage in culture, DM myoblasts show cytoplasmic vacuolization (arrows in c) which become prominent at late passages (d). Bar: 20 µm


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Figure 6. Transmission electron micrographs of myoblasts from healthy (a) and DM2 (b) patients at the second culture passage; conventional ultrastructural morphology (glutaralaldehyde-fixation, OsO4 post-fixation, Epon embedding), lead citrate staining. The myoblast in a) shows a nucleus with scarce heterochromatin, rough endoplasmic reticulum (arrowheads) and a well developed Golgi complex (arrow). The myoblast in b) shows a nucleus with evident heterochromatin (asterisks) and cytoplasmic vacuoles (v). Nu, nucleolus. Bars: 1 Âľm.

CONCLUDING REMARKS Based on the cytochemical and ultrastructural evidence, the skeletal muscle of DM patients seems to share intriguing similarities with the muscle from aged mammals, with special reference to the alterations in the nuclear RNP-containing structures involved in pre-mRNA transcription and splicing. This opens interesting perspectives on the role of the RNP nuclear components in the onset of muscle cell dysfunctions and encourages comparative studies aimed at detecting common cellular mechanisms at the basis of skeletal muscle wasting.

ACKNOWLEDGEMENTS We thank Prof. Giovanni Meola and his collaborators for providing us with muscle biopsies and for helpful discussion. M.G. is a PhD student in receipt of a fellowship from the Dottorato di Ricerca in Biologia Cellulare (University of Pavia).


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Insulin receptor splicing alteration in myotonic dystrophy type 2. Am J Hum Genet 74:1309-13. Schoser BGH, Schneider-Gold C, Kress W, Go Ebel HH, Toyka KV, Lochmuller H, Ricker K, 2004. Muscle pathology in 57 patients with myotonic dystrophy type 2. Muscle Nerve 29:275-81. Schul W, Groenhout B, Koberna K, Takagaki Y, Jenny A, Manders EM, Raska I, van Driel, R, de Jong L, 1996. The RNA 3’ cleavage factors CstF 64 kDa and CPSF 100 kDa are concentrated in nuclear domains closely associated with coiled bodies and newly synthesized RNA. Embo J 15:2883-92. Taneja KL, McCurrach M, Schalling M, Housman D, Singer RH, 1995. Foci of trinucleotide repeat transcripts in nuclei of myotonic dystrophy cells and tissues. J Cell Biol 128:995-1002. Thompson LD, 2009. Age-related muscle dysfunction. Exp Gerontol 44:106-11. Vihola A, Bassez G, Meola G, Zhang S, Haapasalo H, Paetau A, Mancinelli E, Rouche A, Hogrel JY, Laforêt P, Maisonobe T, Pellissier JF, Krahe R, Eymard B, Udd B, 2003. Histopathological differences of myotonic dystrophy type 1 (DM1) and PROMM/DM2. Neurology 60:1854-7. Wahle E, Rüegsegger U, 1999. 3’-end processing of pre-mRNA in eukaryotes. FEMS Microbiol Rev 23:277-95. Wheeler TM, Thornton CA, 2007. Myotonic dystrophy: RNA-mediated muscle disease. Curr Opin Neurol 20:572-6. Zancanaro C, Malatesta M, Vogel P, Osculati F, Fakan S, 1993. Ultrastructural and morphomentrical analyses of the brown adipocyte nucleus in a hibernating dormouse. Biol Cell 79:55-61.


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CLINICAL ASPECTS AND MANAGEMENT OF MYOTONIC DYSTROPHIES GIOVANNI MEOLA (*)(**), ENRICO BUGIARDINI (*), LAURA V. RENNA (**), FRANCESCA RIZZI (**), ROSANNA CARDANI (**)

RIASSUNTO. – La distrofia miotonica (DM) è la più comune forma di distrofia muscolare dell’adulto ad ereditarietà autosomica dominante caratterizzata da miopatia progressiva, miotonia e da un coinvolgimento multisistemico. Ad ora sono state identificate due forme distinte di DM causate da mutazioni simili. La distrofia miotonica di tipo 1 (DM1, malattia di Steinert) è stata descritta più di 100 anni fa ed è causata dall’espansione della tripletta (CTG)n nel gene DMPK, mentre la distrofia miotonica di tipo 2 (DM2) è stata identificata solo 18 anni fa ed è causata dall’espansione (CCTG)n nel gene ZNF9/CNBP. I trascritti mutanti contenenti le espansioni CUG o CCUG, si aggregano sottoforma di foci nei nuclei delle cellule dove sequestrano proteine RNA-binding con conseguente alterazione dello splicing alternativo (spliceopatia) di geni effettori a valle. Nonostante le somiglianze cliniche e genetiche, la DM1 e la DM2 sono disordini ben distinti che richiedono differenti strategie diagnostiche e di gestione. La DM1 può presentare quattro forme clinicamente diverse: la forma congenita, la forma infantile, la forma a esordio adulto e quella ad insorgenza tardiva oligosintomatica. La DM1 congenita è la forma più grave di DM caratterizzata da estrema debolezza muscolare e ritardo mentale. Nella DM2 il fenotipo clinico è molto variabile e non ci sono sottogruppi clinici distinti. Forme congenite e a esordio infantile non sono state descritte nella DM2 e, contrariamente alla DM1, la miotonia può essere assente anche all’esame elettromiografico. A causa della mancanza di conoscenza della malattia tra i medici, la DM2 rimane ampiamente sottodiagnosticata. Il ritardo nel ricevere la diagnosi corretta dopo l’insorgenza dei primi sintomi è molto lungo nelle DM: in media più di 5 anni per la DM1 e più di 14 anni per i pazienti con DM2. Il lungo ritardo nella diagnosi delle DM causa nei pazienti problemi nella ge-

(*)

Department of Neurology, University of Milan, IRCCS Policlinico San Donato, Via Morandi, 30, 20097 San Donato Mil. Milan (Italy). Tel.: +39.02.52774480 Fax: +39.02.5274717. E-mail: giovanni.meola@unimi.it (**) Laboratory of Muscle Histopathology and Molecular Biology, IRCCS Policlinico San Donato, University of Milan, Italy.


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stione delle loro vite e angosce a causa dell’’incertezza della prognosi e del trattamento terapeutico. *** ABSTRACT. – Myotonic dystrophy (DM) is the most common adult muscular dystrophy, characterized by autosomal dominant progressive myopathy, myotonia and multiorgan involvement. To date two distinct forms caused by similar mutations have been identified. Myotonic dystrophy type 1 (DM1, Steinert’s disease) was described more than 100 years ago and is caused by a (CTG)n expansion in DMPK, while myotonic dystrophy type 2 (DM2) was identified only 18 years ago and is caused by a (CCTG)n expansion in ZNF9/CNBP. When transcribed into CUG/CCUG-containing RNA, mutant transcripts aggregate as nuclear foci that sequester RNA-binding proteins, resulting in spliceopathy of downstream effector genes. Despite clinical and genetic similarities, DM1 and DM2 are distinct disorders requiring different diagnostic and management strategies. DM1 may present four different forms: congenital, early childhood, adult onset and late-onset oligosymptomatic DM1. Congenital DM1 is the most severe form of DM characterized by extreme muscle weakness and mental retardation. In DM2 the clinical phenotype is extremely variable and there are no distinct clinical subgroups. Congenital and childhood-onset forms are not present in DM2 and, in contrast to DM1, myotonia may be absent even on EMG. Due to the lack of awareness of the disease among clinicians, DM2 remains largely underdiagnosed. The delay in receiving the correct diagnosis after onset of first symptoms is very long in DM: on average more than 5 years for DM1 and more than 14 years for DM2 patients. The long delay in the diagnosis of DM causes unnecessary problems for the patients to manage their lives and anguish with uncertainty of prognosis and treatment. KEY WORD. – Myotonic dystrophy type 1 (DM1); myotonic dystrophy type 2 (DM2); clinical findings; muscle biopsy; management.

INTRODUCTION Myotonic dystrophies (DMs) are autosomal dominant, multisystemic diseases with a core pattern of clinical presentation including myotonia, muscular dystrophy, cardiac conduction defects, posterior iridescent cataracts, and endocrine disorders (Harper, 2001) . In 1909 Steinert and colleagues first clearly described the “classic” type of myotonic dystrophy which was called Steinert’s disease (OMIM 160900). The gene defect responsible for myotonic dystrophy described by Steinert was discovered in 1992 and found to be caused by expansion of a CTG repeat in the 3’ untranslated region of DMPK, a gene encoding a protein kinase (Brook et al., 1992; Fu et al., 1992,


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Mahadevan et al., 1992). Subsequently, in 1994, a different multisystemic disorder was described with dominantly inherited myotonia, proximal greater than distal weakness, and cataracts but lacking the gene defect responsible for Steinert’s disease (Thornton et al., 1994a; Ricker et al., 1994; Udd et al., 1997). In Europe, the disease was termed proximal myotonic myopathy (PROMM, OMIM*160900) (Ricker et al., 1994) or proximal myotonic dystrophy (PDM) (Udd et al., 1997) while in the United States was termed myotonic dystrophy with no CTG repeat expansion or myotonic dystrophy type 2 (DM2) (Thornton et al., 1994a). Later studies demonstrated that many of the families identified as having DM2, PROMM or PDM had the same disease, a disorder caused by an unstable tetranucleotide CCTG repeat expansion in intron 1 of CCHC-type zing finger, nucleic binding protein (CNBP) mapped to 3q21.3 (Ranum et al., 1998; Liquori et al., 2001). Due to the existence of different types of myotonic dystrophy, the International Myotonic Dystrophy Consortium developed a new nomenclature and guidelines for DNA testing (Ashizawa and Baiget, 2000). The Steinert’s disease, the classic form of myotonic dystrophy that results from an unstable trinucleotide repeat expansion on chromosome 19, is now termed myotonic dystrophy type 1 (DM1). Patients with the clinical picture of DM2/proximal myotonic myopathy, who have positive DNA testing for the unstable tetranucleotide repeat expansion on chromosome 3, are now classified as having myotonic dystrophy type 2 (DM2) (Thornton et al., 1994a; Day et al., 1999; Day et al., 2003). Although DM1 and DM2 have similar symptoms, they also present a number of very dissimilar features making them clearly separate diseases (Table 1).

MYOTONIC DYSTROPHY TYPE 1 CLINICAL FEATURES DM1 is the most common inherited muscular dystrophy in adults with an estimated prevalence of 1:8,000. DM1 is characterized by the phenomenon of anticipation, by which the disease has an earlier onset and more severe course in subsequent generations. Patients with DM1 can be divided into four main categories, each presenting specific clini-


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cal features and management problems: (1) congenital, (2) childhoodonset, (3) adult-onset, and (4) late-onset/asymptomatic. Table 2 summarises these subtypes. Table 1. Comparison of clinical manifestations between DM1 and DM2. Clinical Features

DM1

DM2

General features Epidemiology Age of onset (years) Anticipation Congenital form Life expectancy

Widespread 0 to adult Always present Present Reduced

European ago-60 Exceptional Absent Normal range

Core features Clinical myotonia EMG myotonia Muscle weakness Cataracts

Evident in adult- onset Always present Disabling at age 50 Always present

Present in <50% Absent or variable in many Onset after age 50-70 Present in minority

Muscle symptoms Facial and jaw weakness Always present Bulbar weakness-dysphagia Always later Respiratory muscles weakness Always later Distal limb muscle weakness Always prominent Proximal limb muscle weakness May be absent Sternocleidomastoid weakness Always prominent Myalgic pain Absent or mild Visible muscle atrophy Face, temporal, distal hands and legs Calf hypertrophy Absent

Usually absent Absent Exceptional Only flexor digitorium profundus, rare Main disability in most patients, late Prominent in few Most disabling symptom in many Usually absent Present in ≥50%

Systemic features Tremors Behavioral change Cognitive disorders Hypersomnia Cardiac arrhythmias Male hypogonadism Manifest diabetes

Prominent in many Not apparent Not apparent Infrequent From absent to severe Subclinical in most Infrequent

Absent Early in most Prominent Prominent Always present Manifest Frequent

Table 2. Summary of myotonic dystrophy type 1 phenotypes, clinical findings and CTG length. Phenotypes

Clinical findings

CTG length

Age of onset

Congenital

Infantile hypotonia Respiratory failure Learning disability Cardiorespiratory complications

>1000

Birth

Childhood onset

Facial weakness Myotonia Low IQ Conduction defects

100-1000

1-10 years

Adult onset “classic DM1”

Weakness Myotonia Cataracts Conduction defects Insulin resistance Respiratory failure

100-1000

10-30 years

Late onset/Asymptomatic

Mild myotonia Cataracts

50-100

20-70 years

Pre-mutation

None

38-49

N/A


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CONGENITAL DM1 Congenital DM1 (CDM) shows a distinct clinical phenotype with distinct clinical features therefore it is to be considered a severe early form of ‘classical’ DM1. CDM often presents before birth as polyhydramnios and reduced fetal movements. After delivery, the main features are severe generalized weakness, hypotonia and respiratory compromise. In up to 50% of CDM, bilateral talipes and other contractures are present at birth. One feature of affected infants is the “fish-shaped” upper lip an inverted V-shaped upper lip which is characteristic of severe facial weakness and causes weak cry and the inability to suck. Mortality from respiratory failure is high. Surviving infants experience gradual improvement in motor function, they can swallow and independently ventilate. Almost all CDM children are able to walk. Cognitive and motor milestones are delayed and all patients with CDM develop learning difficulties and require special needs schooling. Cerebral atrophy and ventricular enlargement are often present at birth (Ashizawa, 1998; Spranger et al., 1997). A progressive myopathy and the other features seen in the classical form of DM1 can develop although this does not start until early adulthood and usually progresses slowly (Joseph et al., 1997). Despite the severe muscular phenotype, clinical myotonia is neither a feature presented in the neonatal period nor can it be disclosed in the electromyogram (EMG). Patients often develop severe problems from cardiorespiratory complications in their third and fourth decades. CHILDHOOD ONSET DM1 The diagnosis of this form of DM1 is often missed in affected adolescents or children because of uncharacteristic symptoms for a muscular dystrophy and apparently negative family history (Harper et al., 2002). Cases of DM1 that come to medical attention during childhood typically manifest developmental abnormalities that are less severe than seen in congenital onset cases (O’Brien and Harper, 1984). Unlike the CDM patients, in which maternal transmission is the rule, the sex of the parents does not influence the development of childhood onset DM1. These patients have cognitive deficits and learning abnormalities (Steyaert et al., 2000). As in the congenital cases, degenerative features often develop as these children reach adulthood. There is increasing evidence of early conduction abnormalities, and from the age of 10,


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annual electrocardiograms and consideration of electrophysiological studies should be a part of routine management. ADULT ONSET DM1 The core features in classic DM1 are distal muscle weakness, leading to difficulty with performing tasks requiring fine dexterity of the hands and foot drop, and facial weakness and wasting, giving rise to ptosis and the typical myopathic or â&#x20AC;&#x2DC;hatchetâ&#x20AC;&#x2122; appearance. The neck flexors and finger/wrist flexors are also commonly involved. Grip and percussion myotonia are regular features; however, myotonia affects other muscle including bulbar, tongue or facial muscles, causing problems with talking, chewing, and swallowing. Elevation of the serum creatine kinase is present. Cardiac involvement is common in DM1 and includes conduction abnormalities with arrhythmia and conduction blocks contributing significantly to the morbidity and mortality of the disease (Bassez et al., 2004; Chebel et al., 2005; Montella et al., 2005; Dello Russo et al., 2006). In some patients and families, a dilated cardiomyopathy may be observed. Posterior subcapsular cataracts develop in most patients and some patients may develop cataract at an early age without any other symptoms and then develop muscle symptoms later in their disease (Garrot et al., 2004). Minor intellectual deficits are present in many patients in contrast with CDM and childhood onset DM1. Avoidant, obsessive-compulsive and passive-aggressive personality features have also been reported (Delaporte, 1998; Winblad et al., 2005). Nocturnal apnoeic episodes and daytime sleepiness are a common manifestation. Gastrointestinal tract involvement covers irritable bowel syndrome, symptomatic gall stones and gamma-glutamyltransferase elevations. Finally, endocrine abnormalities include testicular atrophy, hypotestosteronism, insulin resistance with usually mild type-2 diabetes. LATE-ONSET/ASYMPTOMATIC DM1. In late-onset or asymptomatic patients (with low number of CTG repeats), only limited features are found on clinical and paraclinical assessment. Myotonia, weakness and excessive daytime sleepiness are rarely present. Before DNA tests became available, there were many examples of incorrect ascertainment, even when using markers such as EMG evidence of myotonia and slit-lamp examination for the charac-


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teristic cataracts (Barnes et al., 1994). In late-onset patients, the search for cataracts is helpful for identifying the transmitting person.

MYOTONIC DYSTROPHY TYPE 2 CLINICAL FEATURES The prevalence of DM2 is not well established, but estimated to be similar to DM1 in European populations (Udd et al., 2006) In DM2 there are no distinct clinical subgroups although initially, different phenotypes of DM2 were described: DM2/PROMM and PDM (Thornton et al., 1994; Ricker et al., 1994; Udd et al., 1997). The most important discrepancy between DM1 and DM2 is absence of a congenital or earlyonset form in DM2 (Udd et al., 2003; Day et al., 2003) and the clinical presentation is a more continuum from early adult-onset severe form to very lateâ&#x20AC;&#x201C;onset mild symptoms (paucisymptomatic). Clinically based ascertainment of DM2 patients is even more difficult because of the large phenotypic variability and a large number of individuals with milder symptoms who remain undiagnosed. Moreover, milder phenotypes with prominent myalgia may easily be misdiagnosed as fibromyalgia (Auvinen et al., 2008) and patients with onset of slowly progressive proximal muscle weakness after age 70 years may not be referred for neuromuscular investigations. Further evidence that a large proportion of DM2 patients may be undiagnosed came from a recent study which indicate that cosegregation of heteroxygous recessive mutations in chloride channel 1 (CLCN1) gene in DM2 patients is higher than expected (Suominen et al., 2008). In DM2 patients with cosegregating CLCN1 the severity of myotonia appear to be more evident either clinically or on EMG thus these patients could be more easily identified and diagnosed than DM2 patients without the modifier allele. Consequently the majority of DM2 patients remains undiagnosed even in clinical centers with considerable experience with DM2. DM2/PROMM typically appears in adult life and has variable manifestations, such as early-onset cataracts (younger than 50 years), varying grip myotonia, thigh muscle stiffness, and muscle pain, as well as weakness (hip flexors, hip extensors, abdominal muscles, or long flexors of the finger muscles) (Thornton et al., 1994a; Ricker et al., 1994; Ricker et al., 1995; Day et al., 1999; Meola, 2000; Moxley et al.,


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2002; Day et al., 2003; Schoser et al., 2004a). These complaints often appear between 20 and 70 years of age, and patients as well as their care providers ascribe them to overuse of muscles, “pinched nerves,” “sciatica,” arthritis, fibromyalgia, or statin use (George et al., 2004). Early in the presentation of DM2 there is only mild weakness of hip extension, thigh flexion, and finger flexion. Myotonia of grip and thigh muscle stiffness varies from minimal to moderate severity over days to weeks. Myotonia is often less apparent in DM2 compared with patients with DM1. It is more difficult to elicit myotonia on standard EMG testing in DM2 compared to DM1 except for proximal muscles such as the tensor fascia lata and vastus lateralis muscles. In cases of late-onset DM2, myotonia may only appear on electromyographic testing after examination of several muscles (Meola, 2000). Facial weakness is mild in DM2 as muscle wasting in the face and limbs. The cataracts in DM2 have an appearance identical to that observed in DM1 and develop before 50 years of age as iridescent, posterior capsular opacities on slit-lamp. Cardiac problems appear to be less severe and frequent in patients with DM2 than in patients with DM1(Meola et al., 2002; Flachenecker et al., 2003). In DM2, cardiac conduction alterations are primarily limited to first-degree atrio-ventricular and bundle branch block. However, sudden death, pacemaker implantation, and severe cardiac arrhythmias have been described in small numbers of patients (Moxley et al., 2002; Schoser et al., 2004b). In DM2, no ventilatory insufficiency has been reported. Central nervous system involvement represents one of the major differences between DM1 and DM2. Although retarded DM2 individuals have been reported, these occurrences may be either accidental or an infrequent disease consequence (Ricker et al., 1995; Day et al., 2003). The type of cognitive impairment that occurs in DM2 is similar to but less severe than that of DM1. Other manifestations, such as hypogonadism, glucose intolerance, excessive sweating, and dysphagia, may also occur and worsen over time in DM2 (Thornton et al., 1994a; Day et al., 1999; Meola et al., 1999; Newman et al., 1999; Savkur et al., 2001; Day et al., 2003; Meola et al., 2003; Schoser et al., 2004a; Savkur et al., 2004). Pregnancy and menses may also exacerbate muscle pain, myotonia, and muscle cramps (Rudnik-Schoneborn et al., 2006). PDM patients show many features similar to those found in PROMM, including proximal muscle weakness, cataracts, and electrophysiologically detectable myotonia. Unlike PROMM patients, however, they do not report myalgias, symptomatic myotonia, or muscle stiffness. Instead


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they present traits not present in PROMM, such as pronounced dystrophic-atrophic changes in the proximal muscles and late-onset progressive deafness (Udd et al., 1997).

GENETICS The DM1 mutation was identified in 1992 as an expansion of an unstable CTG trinucleotide repeat in the 3’untranslated region (UTR) of the myotonic dystrophy protein kinase gene (DMPK; OMIM 605377) which codes for a myosin kinase expressed in skeletal muscle. The gene is located on chromosome 19q13.3 (Fu et al., 1992; Mahadevan et al., 1992). In DM1 patients the repeat size range from 50-4.000 (150-12.000 bp) and is nearly always associated with symptomatic disease although there are patients who have up to 60 repeats who are asymptomatic into old age and similarly patients with repeat sizes up to 500 who are asymptomatic into middle age. Normal individuals have between 5 and 37 CTG repeats. Patients with between 38 and 49 CTG repeats are asymptomatic but are at risk of having children with larger, pathologically expanded repeats (Thornton et al., 1994a). This is called a ‘pre-mutation’ allele. The DM1 mutation length predicts the clinical outcome to some extent: classical DM1 100-1.000 repeats; congenital >1.000 repeats (Ashizawa and Baiget, 2000; Schoser and Timchenko, 2010). DM2 results from an unstable tetranucleotide repeat expansion, CCTG, in intron 1 of the nucleic acid-binding protein (CNBP) gene (previously known as zinc finger 9 gene, ZNF9) on chromosome 3q21 (Ranum et al., 1998; Liquori et al., 2001). The size of the CCTG repeat is below 30 repeats in normal individuals while the range of expansion sizes in in DM2 patients is huge. The smallest reported mutation vary between 55-75 CCTG (Liquori et al., 2001; Bachinski et al., 2009) and the largest expansions have been measured to be up about 11.000 repeats (Liquori et al., 2001). Both DM1 and DM 2 mutations show instability with variation in different tissue and cell types causing somatic mosaicism (Lavedan et al., 1993; Monckton et al., 1995). The size of the CTG and CCTG repeat appear to increase over time in the same individual, and are dynamic gene defects (Day et al., 2003). However in DM1 children may inherit repeat lengths considerably longer than those present in the transmitting parent. This phenomenon causes anticipation, which is the occurrence of increasing disease severity and decreasing age of onset in successive generations. A child with congenital DM1


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almost always inherits the expanded mutant DMPK allele from their mother. However anticipation may be seen in patients with DM1 who inherit a smaller expanded CTG repeat from their father (Brunner et al., 1993; de Die-Smulders and Smeets, 1997). In DM2 the mutation usually contracts in the next generation being shorter in the children (Day et al., 2003). This may explains some distinct features of DM2 such as the missing of a congenital form, the lack of anticipation and the later onset (Udd et al., 2003). The size of CCTG repeat expansion in leukocyte DNA in DM2 seems to relate in large part to the age of the patient and not necessarily to the severity of symptoms or manifestations. This complicates attempts to correlate the size of the repeat with earlier clinical onset of more severe symptoms as occurs in patients with DM1. However due to somatic mosaicism, CTG repeat size correlates more significantly with age of onset and disease severity below 400 CTG repeats (Hamshere et al., 1999). The correlation between CTG repeat size and the severity of the disease can be observed in blood but not in other organs (eg, muscle). In DM1 the repeat lengths in muscle are shown to be larger (Thornton et al., 1994b) and there is no correlation between the size of the CTG repeats in muscle and the degree of weakness. It should be noted that in clinical practice, the CTG expansion is measured in blood and there is no additional clinical advantage of measuring repeat size in muscle.

MOLECULAR PATHOMECHANISM As described above the two types of the disease are associated with two different loci: DM1 is caused by the expansion of an unstable CTG trinucleotide repeat in the 3 UTR of the DMPK gene (Brook et al., 1992; Mahadevan et al., 1992) while DM2 mutation consists in the expansion of an unstable CCTG tetranucleotide within the first intron of the nucleic acid-binding protein (CNBP) gene (previously known as zinc finger 9, ZNF9) (Liquori et al., 2001).The fact that two repeat sequences located in entirely different genes can cause such similar disease features implies a common pathogenic mechanism. It is now clear that the gain-of-function RNA mechanism is the predominant cause of pathogenesis of myotonic dystrophies in which the expansion mutation, (CTG)n in DM1 and (CCTG)n in DM2, is transcribed and the mutant RNAs containing the repeat expansions accumulate in the cell nuclei as foci, called ribonuclear inclusions, and are responsible for the pathologic features common to


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both disorders. The expanded CUG/CCUG-containing transcripts form hairpins, imperfect double-stranded structure which lead to deregulation of two important RNA-binding proteins, muscleblindâ&#x20AC;&#x201C;like protein 1 (MBNL1) and CUGBP/Elav-like family member 1 (CELF1). In DM1, MBNL1 protein is depleted from the nucleoplasm through recruitment into ribonuclear foci (Jiang et al., 2004; Lin et al., 2006; Mankodi et al., 2005) while CELF1 stabilization by PKC phosphorylation results in increased steady-state levels and protein upregulation (KuyumcuMartinez et al., 2007). A combined effect of decreased MBNL1 and increased CELF1 activity lead to misregulated alternative splicing and other changes of the muscle transcriptome (Ranum and Cooper, 2006; Salisbury et al., 2009). The alteration of pre-mRNA processing strengthens the hypothesis of a spliceopathy which leads to inappropriate expression of embryonic splicing isoforms in adult tissues (Osborne and Thornton, 2006). In DM2, splicing abnormalities are also associated with the sequestration of MBNL1 protein by expanded transcripts (Fardaei et al., 2002; Ranum and Cooper, 2006). However evidence that CELF1 upregulation also occurs in DM2 is conflicting (Lin et al., 2006; Pelletier et al., 2009; Salisbury et al., 2009). Recent data demonstrate that MBNL1-containing foci in DM2 cells also sequester snRNPs and hnRNPs, splicing factors involved in the early phases of transcript processing, thus strengthening the hypothesis that a general alteration of premRNA post-transcriptional pathway could be at the basis of the multifactorial phenotype of DM2 patients (Fakan, 1994; Perdoni et al., 2009). Misregulation of alternative splicing plays a central role in the development of important DM symptoms (Ranum and Cooper, 2006; Osborne and Thornton, 2006). For example, among the skeletal symptoms of DM, myotonia, insulin resistance and cardiac problems are correlated with the disruption of the alternative splicing of the muscle chloride channel CLCN1, of the insulin receptor (IR) and of the cardiac troponin T (TNNT3), respectively (Philips et al., 1998; Savkur et al., 2001; CharletB et al., 2002; Mankodi et al., 2002; Savkur et al., 2004). However, spliceopathy may not fully explain the multisystemic disease spectrum. The underlying mechanism responsible for muscle weakness and wasting remains to be established. Recent findings suggest that DM mutations can affect gene expression in multiple ways. Altered activity and/or localization of MBNL1 and CELF1 may alter transcription, translation and cell signaling (Pascual et al., 2006; Barreau et al., 2006). Moreover it has been demonstrated that in DM1 the highly regulated pathways of miRNA is


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altered in skeletal muscle and heart tissue potentially contributing to DM1 pathogenetic mechanisms (Gambardella et al., 2010; Perbellini et al., 2011; Rau et al., 2011). Another open question in the field of DM is to clarify the pathomecanisms underlying the phenotypic differences between DM1 and DM2. Clinical signs in DM1 and DM2 are similar, but there are some distinguishing features: DM2 is generally less severe and lacks a prevalent congenital form. This suggests that other cellular and molecular pathways are involved besides the shared toxic-RNA gain of function hypothesized. Disease-specific manifestations may result from differences in spatial and temporal expression patterns of DMPK and CNBP genes. Similarly, changes in the expression of neighbouring genes may define disease-specific manifestations. Importantly, the role of CELF1 in DM2 is particularly intriguing with contradictory results being reported (Lin et al., 2006; Salisbury et al., 2009; Pelletier et al., 2009). Another possible explanation for the clinical differences between the two DM forms is the reduction of DMPK or ZNF9/CNBP protein levels in DM1 and DM2 respectively (Fu et al., 1992; Maeda et al., 1995; Huichalaf et al., 2009; Raheem et al., 2010). Indeed both knockout mouse models for DMPK and ZFN9/CNBP show the phenotypic aspects of DM (Reddy et al., 1996; Chen et al., 2007). Taken together these observations seem indicate that the emerging pathways of molecular pathogenesis are far more complex than previously appreciated.

DIAGNOSTICS LABORATORY TESTS As for all genetics diseases with identified mutation, the typical DM1 and DM2 diagnostic method is mutation verification by genetic tests. In the case of DM1, symptoms and family history are often clear and distinctive enough to make a clinical diagnosis, and the mutation can be confirmed by PCR and Southern Blot analysis. PCR analysis is used to detect repeat lengths less than 100 and Southern blot analysis to detect larger expansions. Predictive testing in asymptomatic relatives as well as prenatal and preimplantation diagnosis can also be performed. On the contrary, the wide clinical spectrum of DM2 phenotype makes the clinical diagnosis more difficult. Moreover conventional PCR and Southern blot analysis are not adequate for a definitive molecular


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diagnosis in DM2 due to the extremely large size and somatic instability of the expansion mutation (Liquori et al., 2001; Bachinski et al., 2003). The copy number of DM2 CCTG is below 30 in phenotypically normal individuals and up 11.000 in patients (Day and Ranum, 2005). A complex genotyping diagnostic procedure is now commonly used consisting of a three step molecular protocol (Day et al., 2003; Udd et al., 2003): (1) a conventional PCR assay across the mutation locus using probes binding to mutation flanking sequences can be used for mutation exclusion. In all DM2 patients, a single PCR product representing the normal allele can be identified because the DNA polymerase fail to amplify the mutatnt allele due to length and stable secondary structure. All individuals showing two alleles for the marker are excluded from having the DM2 mutation. However, identical allele size on two normal alleles occurs in 12% of the population; (2) all patients appearing to have one allele need further molecular analysis to determine whether or not they carry a DM2 expansion. Because of the incomplete sensitivity of Southern analysis, a DM2 repeat assay (RP-PCR) was developed; (3) the RP-PCR method involves amplifying the CCTG repeat by PCR, and probing the resultant product with an internal probe to assure specificity. The combined use of these methods allows 99% sensitivity and specificity for known expansions. Several alternative and highly sensitive methods have been developed for DM2 mutation verification including long-range PCR (Bonifazi et al., 2004) and a tetraplet-primed PCR (Catalli et al., 2010). A modified Southern method using field â&#x20AC;&#x201C;inversion electrophoresis (FIGE) is particularly efficient in determining the mutation length (Bachinski et al., 2003). However, these methods are still too long and complicated to be part of routine laboratory diagnostics. Nevertheless ribonuclear foci and splicing changes are present before any histological abnormality manifestations (Mankodi et al., 2001; Savkur et al., 2004). This could be important for an early diagnosis before the spectrum of clinical signs of muscle disease appear. So a more practical tool to obtain a definitive DM2 diagnosis in few hours is represented by in situ hybridization (ISH) which is a method that allows the direct visualization of the mutant RNA on muscle biopsy (Cardani et al., 2004; Sallinen et al., 2004). By using specific probes for CCUG expansions, it permits a differential diagnosis between DM2 and DM1. Therefore it may be a simple approach for DM2 diagnosis, which can be performed in a rapid and sensitive manner in any pathology laboratory. ISH with CAGG probe should be considered as a routine laboratory procedure to confirm or refute the clinical


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suspicion of DM2. It should also be applied routinely to screen patients with myotonic disorders (Cardani et al., 2004; Sallinen et al., 2004). This approach makes muscle biopsy an essential tool for DM2 diagnosis (Fig. 1A). Moreover, since MBNL1 is sequestered by mutant RNA foci, it is possible to visualize the nuclear accumulation of MBNL1 by immunofluorescence on muscle sections (Fig. 1B). However, although MBNL1 represents an histopathological marker of DM, it does not allow to distinguish between DM1 and DM2 (Cardani et al., 2006) (Table 3).

Figure 1. Fluorescence in situ hybridization (FISH) in combination with MBNL1-immunofluorence on DM2 muscle section. A. Visualization of (CCTG)n expansion on muscle section by FISH using (CAGG)5 specific probe. Red spots within myonuclei (blue, DAPI) represent ribonuclear inclusions containing accumulated mutant RNAs. B. Visualization of nuclear foci of MBNL1 (green spots) colocalizing with ribonuclear inclusions in A.

Table 3. Muscle histopatology in DM1 and DM2. Histopathological findings

DM1

DM2

Fiber size variation

+++

+++

Internal nuclei

+++

+++ more in type 2 fibers

Type 1 fiber atrophy

++

-

Type 2 fiber atrophy

+

++

Type 2 fiber hypertrophy

-

+

+ at advanced stage only

+++ more in advanced stage

± type 1 and type 2 fibers at advanced stage only

+++ type 2 fibers

Ring fibers

++

+

Sarcoplasmic masses

++

±

Fibrosis

+++ at late stage only

++ at late stage only

Fatty replacement

+++ at late stage only

++ at late stage only

Nuclear clump fibers Atrophic fibers (diam. ≤ 6µm)

+++ present in >75% of biopsies; ++ present in 20-50% of biopsies; + present in 10-24% of biopsies; ± occasionally present; - absent.


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MUSCLE BIOPSY The histological features of muscle in DM1 and DM2 are very similar (Fig. 2), and sufficiently characteristic that a diagnosis of DM can be suggested based on muscle biopsy alone (Harper, 2001; Day et al., 2003; Schoser et al., 2004c). In both diseases, affected muscles show a high number of central nuclei and a markedly increased variation in fiber diameter that commonly ranges from less than 10 Âľm to greater than 100 Âľm (Fig. 2A, D).

Figure 2. Panel showing muscle histology in DM1 and DM2. A-C. Transversal sections from DM1 muscle biopsies. A. Haematoxylin & Eosin: fiber size variation and central nuclei (arrows) are present. B, C. The population of atrophic fibers (white arrow) are preferentially type 1 fibers as demonstrated in sections stained for ATPase pH 4.3 (B, dark brown) or immunostained for myosin MHCslow (C, brown). Black arrow indicate centrally located nuclei. D-F Transversal sections from DM2 muscle biopsies. D. Haematoxylin & Eosin: as in DM1 muscle, fiber size variation and central nuclei (arrows) are present. Abundant nuclear clumps are also present (arrow heads) despite the muscle shows an early stage pathology. E, F. Type 2 fibers are predominantly affected in DM2 muscle: in routine laboratory muscle staining such as ATPase pH 10.0 (E) or immunostaining for myosin MHCfast (F), type 2 fiber atrophy (white arrows) and type 2 central nucleation (black arrow) are commonly observed.


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Basophilic regenerating fibers, splitting fibers, fibrosis and adipose deposition occur in both diseases to a variable degree depending on the extent of muscle involvement. Ring finger fibers and sarcoplasmic masses are generally more frequent in DM1 muscle biopsy. Recently the comparison of muscle biopsy findings in classic DM1 with those in DM2 has indicated that specific features are present in DM2 muscle biopsy helping the diagnosis of DM2. Severely atrophic fibers with pyknotic nuclear clumps similar in appearance to the severely atrophic fibers in neurogenic atrophy are frequently found in DM2 biopsy also before the occurrence of muscle weakness (Fig. 2D). In DM1, nuclear clumps are present in end-stage muscle biopsy (Vihola et al., 2010). A predominant type 2 fiber atrophy in contrast to the type 1 atrophy observed in DM1, has been described in DM2 (Vihola et al., 2003; Schoser et al., 2004c; Bassez et al., 2008; Pisani et al., 2008) (Fig. 2B,C,E,F). Moreover, in DM2 muscle biopsy central nucleation selectively affects type 2 fibers and the atrophic nuclear clumps express fast myosin isoform (type 2 fiber) indicating that DM2 is predominantly a disease of type 2 myofibers (Bassez et al., 2008) (Fig. 2F).

MANAGEMENT In general the management of DM2 is similar to that of DM1, but there is less need for supportive care, such as bracing, scooters, or wheelchairs. Cataracts require monitoring. Cardiorespiratory disorders are responsible for 70% of the mortality in DM1 and many of these patients could have been treated by active monitoring and a lower threshold for input. Disturbances in cardiac rhythm are less frequent in DM2, but abnormalities do occur (Day et al., 2003; Moxley et al., 2000; Flachenecker et al., 2003; Schoser et al., 2004b), and serial monitoring with an electrocardiogram is necessary to check for covert dysrhythmia. Hypogonadism and insulin resistance need monitoring in both diseases. Myotonia tends to be less marked and less troublesome in DM2, but in specific circumstances antimyotonia therapy is helpful, especially if muscle stiffness is frequent and persistent or if pain is prominent (Kwiencinski et al., 1992). Cognitive difficulties also occur in DM2 as in DM1 but become manifest in adult life and appear to be associated with decreased cerebral blood flow to frontal and anterior temporal


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lobes (Meola et al., 1999; Meola and Sansone, 2007) and decreased brain volume (Chang et al., 1998; Akiguchi et al., 1999). The changes are less severe than in DM1. Their etiology is unknown but may relate to the toxic effect of intranuclear accumulations of abnormally expanded RNA. Management of these brain symptoms is similar to that for DM1. A frequent and difficult problem in DM2 is the peculiar muscle pain described earlier (George et al., 2004; Auvinen et al., 2008). The exact mechanism underlying the pain is unknown, and there is no wellestablished, effective treatment. Carbamazepine or mexiletine along with nonsteroidal anti-inflammatory medications or tylenol ameliorate this pain in some patients.

CONCLUDING REMARKS The myotonic dystrophies are dominantly inherited multisystemic disorders that include two genetically distinct types. DM1 is the commonest cause of adult onset muscular dystrophy with an estimated prevalence of 1/8000. Due to the lack of awareness of the disease among clinicians, DM2 remains largely underdiagnosed and the prevalence of DM2 is not well established. These diseases have been called â&#x20AC;&#x2DC;spliceopathiesâ&#x20AC;&#x2122; and are mediated by a primary disorder of RNA rather than proteins, however, spliceopathy may not fully explain the multisystemic disease spectrum. Although the two forms of myotonic dystrophy share many features, there are definite differences with respect to clinical, muscle biopsy, and genetic findings. In DM2 the core symptoms include proximal muscle weakness, myotonia, cataracts, cardiac conduction defects, insulin resistance and male hypogonadism. In DM1, the muscle weakness and wasting are more severe, preferentially distal and facial with ptosis, and with later evolving dysphagia, generalized weakness and respiratory failure. A severe congenital form associated with DM1 has not been observed in DM2, and anticipation is the exception in DM2. In contrast to DM1, type 2 fiber are preferentially involved in DM2 with the presence of very atrophic type 2 fibers early in muscle pathogenesis. The basis for the differences between DM1 and DM2 has not been clarified at the molecular level. There is currently no cure but effective management is likely to significantly reduce the morbidity and mortality of patients. The enormous advances in the understanding of the molecular pathogenesis of DM1 and DM2 has revealed pathways of


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molecular pathogenesis more complex than previously appreciated that could be the right track towards the development of effective therapies.

AKNOWLEDGMENTS This work was supported by AFM – Association Française contre les Myopathies, CMN – Centro per lo Studio delle Malattie Neuromuscolari and FMM-Fondazione Malattie Miotoniche.


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INDEX

GIANPIERO SIRONI: Saluto del Presidente dellâ&#x20AC;&#x2122;Istituto Lombardo . . . . . . . . . . . Pag. 3 CARLO PELLICCIARI: Myotonic dystrophies: genetically-based diseases due to toxic RNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5

DENIS FURLING: Splicing abnormalities in myotonic dystrophies . . . . . . . . . . . .

9

MANUELA MALATESTA, MARZIA GIAGNACOVO: Cell nuclear alterations in myotonic dystrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

25

GIOVANNI MEOLA, ENRICO BUGIARDINI, LAURA V. RENNA, FRANCESCA RIZZI, ROSANNA CARDANI: Clinical aspects and management of myotonic dystrophies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

41


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