140th ENMC International Workshop: Myotonic Dystrophy DM2/PROMM and other myotonic dystrophies with guidelines on management *

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1 Neuromuscular Disorders 16 (2006) Workshop report 140th ENMC International Workshop: Myotonic Dystrophy DM2/PROMM and other myotonic dystrophies with guidelines on management * B. Udd *, G. Meola, R. Krahe, C. Thornton, L.P.W. Ranum, G. Bassez, W. Kress, B. Schoser, R. Moxley Neurology Department, Tampere University Hospital and Tampere University Medical School, Folkhalsan Institute of Genetics, Department of Medical Genetics, University of Helsinki, and Vasa Central Hospital, Finland Received 9 March 2006; received in revised form 9 March 2006; accepted 16 March 2006 To the memory of Dr Ken Ricker 1. Introduction: Then and Now for myotonic dystrophy type 2 Myotonic dystrophy is the combination of progressive myotonic myopathy, multiorgan involvement and autosomal dominant inheritance. Currently, two distinct entities of myotonic dystrophy are characterized and defined by their underlying molecular genetic cause. Myotonic dystrophy type 1 (DM1, Steinert s disease) is considered to be the most frequent muscular dystrophy in adults [1]. Starting in 1994 publications of another disorder with similar core features but lacking the DM1 mutation have emerged [2,3]. The name first used for this disorder was proximal myotonic myopathy (PROMM, OMIM *160900), and the first ENMC workshop on PROMM in 1997 established its clinical diagnostic criteria [4]. Prior to the second ENMC Workshop on PROMM in 2000, Ranum et al. [5] published mapping of a new genetic locus in a large Minnesota family with myotonic dystrophy-like features on chromosome 3q21. This locus was named DM2. As a result, a new nomenclature emerged and the locus for myotonic * The ENMC consortium on myotonic dystrophy type 2 (ZDM2 /PROMM) held its 4th workshop in Naarden, The Netherlands, 20 22th March It was attended by 21 active participants from Finland, France, Germany, Italy, Spain, and the USA. The workshop opened with a memorial to honor the past pioneer in the field: Dr Kenneth Ricker and his extensive clinical work on DM2 and other myotonic disorders. * Corresponding author. Tel.: C ; fax: C address: bjarne.udd@netikka.fi (B. Udd) /$ - see front matter q 2006 Elsevier B.V. All rights reserved. doi: /j.nmd dystrophy/steinert s disease was renamed DM1 [6]. Subsequent work showed that most PROMM families mapped to the DM2 locus. One separately described family with proximal myotonic dystrophy [7], also mapped to the DM2 locus [8], and the workshop adopted the term myotonic dystrophy type 2 (DM2) for all the progressive myotonic multiorgan disorders linked to the DM2 locus. Prior to the 3rd workshop on DM2/PROMM in 2003, the identification of the mutation underlying DM2 was accomplished [9]. The mutation is a huge (CCTG)n microsatellite repeat expansion in the first intron of the ZNF9 gene and is thus similar to the (CTG)n repeat causing DM1. The clinical course of DM2 is usually more favorable compared to DM1. Families with DM2 do not have the severe congenital form of illness that occurs in DM1. Abnormalities in the social and cognitive abilities of adults with DM2 are typically mild or absent, and there is no prominent late weakness of the respiratory, facial and bulbar muscles [10]. In DM2 the manual skills largely remain intact and complications during general anesthesia have not been reported. However, DM2 may present with very severe variants of the disease: severe fatal cardiac complications have occurred as well as severe muscle weakness and disability [11]. The aims of this 4th ENMC Workshop on DM2/PROMM and other myotonic dystrophies were: (a) to review the origin and population frequencies of DM2 mutation (b) to evaluate the current state of molecular diagnosis in DM2 and the availability of DNA-tests

2 404 B. Udd et al. / Neuromuscular Disorders 16 (2006) (c) to assess the range of clinical symptoms, signs and findings in DM2 mutation verified patients (d) to present new findings on muscle biopsy, imaging and brain examinations in DM2 patients (e) to thoroughly review and discuss the molecular pathomechanisms caused by the DM2 mutation, both in terms of basic aspects of RNA-metabolism and applied data emerging from the study of secondary effects on other genes and large scale expression profiling; (f) to learn about the first DM2 mouse models been generated (g) to clinically and genetically evaluate DM-disorders without DM1/DM2 mutation including a possible locus on chromosome 16p (h) to provide new guidelines for diagnostic DNA-testing and clinical management of DM2 and to make these guidelines easily accessible on the ENMC and corresponding websites. 2. Frequency and origin of the DM2 mutation Ralf Krahe (TX, USA) started the scientific sessions by reporting on DM2 mutation studies in patients and 93 DM2 kindreds of various ethnic populations from Europe and the USA. The mutation itself is a repeat expansion in the microsatellite CL3N58 (from hereon referred to as the DM2 repeat) in the first intron of the ZNF9 gene that has a complex structure: (TG) (TCTG) 4 10 (CCTG) When mutated the (CCTG)n portion of the repeat tract is expanded to repeat lengths approximately between 75 and 11,000 repeats, such that the mutant allele cannot be amplified by PCR. The normal variation of allele sizes of the DM2 locus ranges from 197 to 294 bp in Caucasians and from 197 to 312 bp in sub-saharan Africans. All patients share an identical core haplotype defined by microsatellite repeat markers and a number of single nucleotide polymorphisms (SNPs), indicating a single ancestral founder mutation event dating ,000 years after the migration out of Africa [8]. The DM2 mutation, to date, has not been identified in sub-saharan or East-Asian populations. In different countries patients share extended common haplotypes, such as the Finns, compatible with the known degree of linkage disequilibrium (LD) in the respective populations. Existence and co-segregation of a genomic cis-acting element centromeric to the DM2 locus, which pre-disposed to increased genetic instability at the DM2 repeat, was proposed. Different populations show different extents of LD in the regions centromeric and telomeric of the core haplotype. Consistent with that hypothesis, Caucasians show greater LD in the centromeric region, while non-caucasian populations such as the sub- Saharan and Chinese show no LD, or pronounced LD on the telomeric side. For mutation detection, the Krahe lab uses a combination of genotyping across the normal DM2 repeat to detect two normal sized alleles, and the previously described repeat-primed PCR (RP-PCR) protocol specific for the DM2 repeat [8], which provide high sensitivity and specificity. For accurate sizing by Southern blot, the lab uses their filed inversion gel electrophoresis (FIGE) protocol [8]. Benedikt Schoser (Germany) continued with new epidemiological data collected on myotonic dystrophies in Germany. In the Munich center alone, around new DM2 diagnoses are made each year and the total number of new diagnoses/year in all Germany is in the order of 200. In the genetic lab in Würzburg, 108 new diagnoses were made in 2004 and 95 in During a 3-year period a total of 1137 were tested for DM1 and of these 584 were confirmed with DM1 mutation. In the same period, 685 patients were tested for DM2 out of whom 384 were confirmed with DM2 mutation. Bjarne Udd (Finland) reported surprising large numbers of newly diagnosed DM2 patients in Finland: 56 patients were genetically confirmed in the last 2 years Of these 56 patients, 27 were referred new families because of clinical manifestations, 16 were symptomatic patients in previously identified families and 13 were identified by typical histopathological findings on muscle biopsy. Added to the previously identified 33 patients [11], the current total of 89 patients on a population of 5 Mio people already exceeds previous cautious estimates on DM2 prevalence. In the Central-Finland hospital district with a population of 260,000 people, where clinicians have been alert regarding variable phenotypes of DM2, the current prevalence of mutation verified DM2 patients is around 1/10,000. Considering the fact that many symptomatic relatives in the known families have not yet been tested, it is obvious that the occurrence of DM2 is at least as high as the generally accepted incidence for DM1 1/8000. The final DM2 prevalence may well be higher than DM1. Laura Ranum (MN, USA) described their further studies on the DM2 mutation and new approaches for linkage studies using small families. The DM2 locus has a complex repeat motif with the following configuration (TG) (TCTG) 4 10 (CCTG) [9]. She showed data indicating that all three repeat motifs are unstable at the population level, although only the CCTG portion of the repeat tract expands in affected individuals. The CCTG repeat is usually interrupted, but in the DM2 patients the expanded CCTG repeat is uninterrupted [12]. So far, only one healthy individual has been identified without interruptions in the CCTG repeat tract. Conservation of this normal allele with the rare short-tandem-repeat marker haplotype found in DM2 patients suggests that the loss of interruptions within the CCTG repeat tract may pre-dispose the repeat to expansion [12]. Wolfram Kress (Germany) reviewed the diagnostic procedures used in European labs for routine mutation detection. Currently, five labs in Germany provide service based DM2 mutation analysis. In the UK also, five labs have

3 B. Udd et al. / Neuromuscular Disorders 16 (2006) DM2 mutation detection on the program, and further labs providing genetic analysis are: one in Switzerland, three labs in France, two labs in Italy, one in the Netherlands, one in Belgium, and one lab in Finland. In Würzburg, the total number of mutation verified patients from 2003 through 2005 is 267. During the exact same period of time, the number of diagnosed DM1 patients was 277, suggesting that in Germany, as with Finland, the real prevalence of DM2 could well be the same as for DM1. There is no particular reason to believe the frequency of the mutation would be highly variable in different European populations. Among the detected mutations 15 were children showing the same size of the expansion as their transmitting parent, a result not fully congruent with previous studies indicating expanding size with age. The diagnostic protocol for DM2 used in Würzburg is based on screening/exclusion by DM2 allele genotyping, followed by mutation analysis with long-range PCR- Southern in case of one amplified normal DM2 allele [13]. Even when using three different concentrations this method gives false negative results in 2 8%, but false positive results have not occurred. However, for accuracy, the quality of DNA is crucial. As a second method for mutation detection, Southern blot on Taq1 digested DNA in a routine or pulse field electrophoresis can be used. The quality control of the analyses in different labs was extensively discussed. In France, the three qualified labs have internal controls. At the workshop, the six European and one US (Krahe lab) labs represented agreed on continuously exchanging samples, which prove to be difficult to analyze. In the USA, the commercial Athena lab handles most of the molecular diagnostics. Dr Kress will also discuss with the European EMQN quality network how to integrate the DM2 analytic procedures. 3. The clinical spectrum of symptoms and signs in patients with DM2 mutation Guillaume Bassez (France) presented experience from the testing lab and the clinical setting in Paris. During the last 13 years the average number of DM1 analyses has been 170/year with an average of 75 positive results each year. During the last 3 years the lab has analyzed 98 samples for DM2 mutation out of which 45 were positive. Many of the DM2 negative samples proved later to have CLC1 mutation. The group also analyzed 74 stored biopsies with chromogenic in situ hybridization (CISH) [14] yielding six positive DM2 mutations. Out of 37 referred muscle biopsies for DM2 evaluation 16 were mutation positive. The French DM2 cohort includes patients from Algeria, Lebanon and one with ancestry in Marocco/Senegal. Increasingly, very mild phenotypes were encountered such as, one patient diagnosed at age 49 complaining of myalgia but still above average active in football. Another 47-year-old patient was athletic and participated in marathon runs. His only symptoms were myalgia and an elevated CK of 9000 after an infection but without muscle exercise. Another patient had just exercise myalgia, gynecomastia with low testosteron and a CK of 3! the UNL. The so far latest onset of symptoms, mild lower leg weakness starting at 77, was recorded in a 81-year-old patient. A peculiar finding was vomiting due to pyloric stenosis in one patient. As a cohort 21 patients with camptocormia [15] were analyzed with two DM2 positive results, suggesting DM2 should be considered in this patient group. On the severe end, one 30-year-old pregnant woman had a CK of 700 and severe dilative cardiomyopathy with ejection fraction of 25% and proximal muscle atrophy. Hearing loss has been reported in a few DM2 families, including the original Finnish PDM family. In the French material, considerable hearing loss was present in 5/29 patients compared with 10/25 in DM1 [16]. Satu Auvinen (Finland) reported three highly unusual phenotypes in DM2 verified patients. One was a male patient starting to have spells of periodic paralysis (PP) at the age of 25 years causing repeated visits to the emergency. The patient was extensively studied with exclusion of known causes for PP already 23 years ago. During all the years some EMG recordings showed myotonic features and others did not. The attacks of PP still prevail after DM2 diagnosis at the age of 50 years. Another patient was repeatedly in the emergency because of attacks of dystonia and myoclonus starting at the age of 35 years. Her sister had milder similar symptoms and both had some myotonia on EMG leading to the evaluation of DM2. The third presented patient had onset of dyspnea at the age around 60 years and later developed proximal muscle weakness. However, respiratory failure was the major disability with assisted ventilation at the age of 73 years. Benedikt Schoser presented an update on the rare Afghan family with three homozygous patients [17]. The general aspect that their phenotype does not fall out of range of the wide spectrum of heterozygote phenotypes still holds. Temporal atrophy was prominent but muscle weakness was not more than moderate at ages and with normal CK. However, a daughter had mild proximal weakness and myotonia already at the age of /11 of the heterozygote children have elevated CK and 2/11 have elevated gamma-gt in the same age range. Christiane Schneider-Gold (Germany) described their clinical experience and recent studies on the heart in DM2 by MRI [18,19]. One patient with elevated CK had LBBB at age 23 and progressed to heart failure and manifest dilative cardiomyopathy at age 45 needing a Pacemaker. He had proximal weakness, cataracts, EMG myotonia. Finally, DM2 diagnosis was established at the age of 58. In a series of 11 DM2 patients studied left ventricle volume was increased, while ATP and pcr were decreased without significant differences between younger and older patients. In a retrospective analysis of 297 patient records, four patients had sudden cardiac death by left ventricular

4 406 B. Udd et al. / Neuromuscular Disorders 16 (2006) DCM and severe conduction defects. At least four patients with proven DM2 and dilatative cardiomyopathy underwent heart transplantation prior to DM2 diagnosis. Cornelia Kornblum (Germany) reported the results of whole body MRI of muscles in 15 DM1 and 14 DM2 patients using a 3 T equipment. All DM1 patients have fatty degenerative changes most pronounced in the lower legs where posterior compartment was more severely replaced by fatty and connective tissue than the anterior compartment. In later stages of disease, proximal thigh muscles also were involved many with a characteristic lunar shaped change around the femoral bone due to dystrophic change in vastus intermedius. Some sign of muscle edema was encountered in 30%. In thoracal sections, dilatation of oesophagus was noted in DM1 but not in DM2 patients. DM2 patients had a very different pattern with many almost normal findings despite proximal weakness. Thirty-four percent had changes in erector spinae and gluteus maximus muscles. Christiane Schneider-Gold (Germany) and her group have made systemic studies on myalgia with the conclusion that DM2 patients have different types of pains, myalgia and cramps even in the same patient. Compared with patients having oculopharyngeal dystrophy, DM2 patients have more pronounced localizations in thighs, back more and proximal upper limbs. Another reported unpleasant sensation was not really pain but more like tenderness in the muscles. Symptoms were usually enhanced by cold, static postures and heavier exercise. In 30% of patients, muscle pain was the most disabling symptom and tended to increase with disease duration. Giovanni Meola (Italy) updated their long-term studies on brain functions and abnormalities in DM2 patients [20]. MMSE, particularly visuo-spatial and frontal tests are affected in DM2, whereas IQ is not affected. In personality tests, DM1 patients have a range of changes that DM2 patients do not share, but for some of avoidant character. On MRI DM2 patients have similar but milder white matter changes compared with DM1 in correlation with the results that CSF protein is elevated in 20% of DM1 patients but always normal in DM2. Tremor is a common symptom and hypersomnia occurred in 20% of patients in DM2. They also studied one DM2 patient with camptocormia with PET: mesotemporal glucose was diminished, but the DOPA pathway was normal [21]. In a follow-up of cognitive functions over 7 years the decline of brain abnormalities, although milder in comparison to DM1, was confined to frontal lobe dysfunction in DM2. In keeping with the results published on abnormal splicing of Tau protein in brain tissue of one Finnish DM2 patient [22] similar to DM1 situation, Tau in CSF was elevated in 25% of DM2 patients at comparable levels to those seen in FTD. Anna Vihola (Finland) discussed results of an ongoing project to identify the molecular background and cause of the main muscle pathology feature in DM2: the very small type 2 fibers. These minifibers, nuclear clump fibers and uninucleated fibers, are scattered, occur early even at the time of first symptoms and constitute a subpopulation of type 2 fibers [23]. The concept of the study has been to use immunohistochemistry on proteins known to be expressed in type 2 fibers to identify those differentially expressed in small vs. normal sized type 2 fibers. Markers for development and satellite cells were also used. Results show unexplained preference for neonatal myosin and the anti-apoptotic Bcl-2 to be highly expressed in the small atrophic fibers, which share features with nuclear clump fibers occurring in neurogenic atrophy. 4. Molecular pathomechanisms of DM2 Charles Thornton (USA) started the highly interesting session by discussing the effects of retained mutant ZNF9- transcript and the consequences of Muscleblind (MBLN1, 2 and 3) protein sequestration. MBLN normally binds target RNAs and mediates control of splicing events of premrna. When MBLN is sequestered by mutant (CAGG)n the net effect is loss of MBLN function [24,25]. There are basically two types of RNA-binding proteins: the MBLNfamily and the CUG-BP/CELF-family with different modes of splicing regulation. For the study of the molecular events different transgenic mouse models have been developed. Both the homozygous KI-mouse with a 250 CTG repeat in a human actin construct and the functional Mbln-KO mouse by Mbln exon 3 skipping, show myotonic dystrophy phenotypes and similar abnormal splicing of Clc1 premrna [26]. These models have been further used for screening of other aberrantly spliced genes. These screens have so far confirmed ZASP, SERCA1, TTN and MBLN1 and 2 to be involved. The similar abnormal effects on splicing observed both in the Mbln-KO and the (CTG) TG mice cause a block in the transition to adult isoforms and retention of developmental isoforms of genes. Overall MBLN and CUG-BP protein levels were not altered in neither of the mice models nor in DM muscle in these experiments. However, MBLN in the nucleoplasm was decreased in DM1/DM2 and observed in the ribonuclear foci only and with higher degree of aggregation in DM2 than in DM1. CUG-BP did not co-localize with nuclear ribonuclear inclusions. The double heterozygous (CTG) TG/Mbln-KOG shows electrical myotonia, CLC1 splicing abnormalities and ringfibers in muscle histopathology. Some promise for future therapy may be derived from the observation that MBLN1 gene therapy works both in the (CTG) 250 -TG mouse and in the Mbln-KO mouse to rescue abnormal splicing events. Laura Ranum (USA) continued with highlighting the effects of (CCTG)n expansion on ZNF9 expression and alternative splicing changes in DM2 [27]. The splicing of ZNF9 transcripts seems not to be affected and ZNF9 mrna levels were normal in cultured DM2 myoblasts. Fluorescence in situ hybridization studies and other molecular

5 B. Udd et al. / Neuromuscular Disorders 16 (2006) studies show that the DM2 expansion does not affect normal splicing of intron1 or the export of mature mrna to the cytoplasm. This is consistent with the normal ZNF9 mrna and protein levels found in both heterozygous and homozygous myoblasts and in heterozygous DM2 muscle biopsy tissue. Attempts to correlate expansion sizes with clinical outcome were not possible because of the complexity and heterogeneity of the size of the mutant expansion. The studied homozygous patient showed expansion size of 11,000 repeats in the blood lymphocytes, whereas four lower expansion sizes were encountered in myoblasts. One patient with typical DM2 phenotype and a very low expansion size of 75 CCTG-repeats in blood showed very high instability in muscle with up to 5000 repeats in the mutant expansion. Annalisa Botta (Italy) reported their published studies on ZNF9 expression in DM2 lymphoblasts [28]. Their patient material consisted of four DM2 patients with 15 kb expansions (almost 4000 repeats) in blood lymphocytes. ZNF9 mrna levels both in cytoplasmic and nuclear fractions were normal as measured with an exon1 2 probe. ZNF9 is normally spliced with variant isoforms a, b and g depending on inclusion of intron 2. Specific testing of the a-isoform mrna was not different to controls. Normal function of ZNF9 is binding RNA and interfering with TOP translation. Localization in muscle is intramyofibrillar with a sarcomeric banding similar to SERCA but without co-localization. Protein expression measured by Western blotting showed high variation, without essential difference to the controls. Lubov Timchenko (USA) highlighted the role of protein protein complexes and CUGBP1 in DM [29]. In DM1, mutant (CUG)n transcripts are not only retained in the nucleus but also found in the cytoplasm, whereas the wt DMPK mrna is detected only in the cytoplasm. Immunofluorescence studies on CUGBP1 show co-localization with DMPK transcript in the cytoplasm and in the nuclei of DM1 muscle. Targets for CUGBP1 mediated modification of translation are MEF2A, CEBPB and p21 mrnas encoding proteins involved in the control of myogenesis. CUGBP1 is clearly involved in the abnormal splicing events seen in DM (e.g. ctnt, CLC1 and IR). Abnormal translation and splicing mediated by CUGBP1 are associated with increased levels of CUGBP1 in DM. Studies have revealed at least two new types of mega protein protein complexes (MPPC1 and 2) that are increased in DM2 with both CUGBP1 and CCUG-RNA as parts of the complexes. The MPPC1 complex has been purified and individual proteins of MPPC1 have been identified: er99, Grp78, er60, CRT, eif2b, eif2a, eifg (SR and ER chaperons, proteins for translation initiation). Isolation of biologically active MPPC1 has shown that several components of the complex are abnormally phosphorylated. Results also indicate an increased translation of MEF2A, p21 and truncated isoform of C/EBP, LIP, which is consistent with these proteins being increased in DM2. The level of phosphorylated CUGBP1 is increased in DM2 cells. The inhibition of CUGBP1 by sirna silencing causes decrease of MPPC1. Another complex MPPC2 contains some 10 proteins, some of which also occur in MPPC1. Taken together, CUGBP1 may lead to additional abnormalities of protein isoform expression in DM2 by interference on the translational level. Karin Jurkat-Rott (Germany) reported their extensive studies on the splicing abnormalities of the CLCN1 gene and effects on myotonia. Five splice variants were identified: exon2-, exon9-, exon11-, exons (2 12)- and exons (6 7). The last isoform constitutes 70 80% of chloride channel protein in DM2. However, even in normal controls less than 50% is full length protein. The exon (6 7) isoform is not capable of inducing any Cl-current, and a mix of 50 wt% protein results in lower than half of the normal current. Even transient expression of low repeats (CCUG) 18 and (CUG) 24 in C2C12 cell lines caused abnormal splicing 1 out of 20 experiments never seen in controls. In their studies not only CLC1 underwent abnormal splicing but also sodium- and calcium-channels. Charles Thornton (USA) and his group have analyzed the relative proportions of abnormal splicing or reduced transcription as the cause of reduced CLC1 expression. Stop codons in CLC1 have been shown to cause reduced mrna due to nonsense mediated decay, and CLC1 undergoes a complex transition of isoforms during development with adult isoforms increasing the Cl-current. The transitions are RUST-regulated (Regulation by Unproductive Splicing and Translation), and correlated with development of T-tubule system. Calculations based on known steady state 1 2 -lives of transcripts indicate that abnormal splicing is sufficient to explain the low levels of CLC1 mrna in DM patients. In accord with these calculations abnormally spliced CLC1 is 24% in control vastus lateralis muscle, 50% in mild DM1, 80% in severe DM1, and 90% in DM2 muscle. The group did not observe abnormal splicing of sodium channel and the question why EMG myotonia is more pronounced in DM1 compared to DM2 is not fully answered by known abnormalities in CLC1. However, the T-tubule portion of CLC1 may be more important than the sarcolemmal portion, and many other channels may have important roles considering that the proportions of different currents are the key issue for myotonia. Laura Ranum (USA) summarized recent work on the development of a mouse model for spinocerebellar ataxia type 8 (SCA8). SCA8 is a CNS disorder related to the myotonic dystrophies because it involves the expression of non-coding CUG expansion transcripts. These animals develop a striking and often fatal CNS phenotype. Molecular and phenotypic characterization is being performed to study possible RNA gain of function mechanisms in the CNS that may parallel those found in DM1.

6 408 B. Udd et al. / Neuromuscular Disorders 16 (2006) Partha Sarkar (USA) presented exciting new data generated in collaboration with T Ashizawa. Accumulation of mutant DM1 (CUG)n containing transcripts result in the hyperactivation of the Notch signaling pathway. By capturing RNA-binding proteins on affinity columns they identified three new Zinc-finger proteins, STR-1, Eth-1, and a third one. STR-1 is apparently involved in Notch1- signalling and is sequestered in the ribonuclear inclusions at least in DM1, but not present in normal C2C12 nuclei. Early results suggest it is not present in the DM2 ribonuclear inclusions. Defects in myogenesis are shown in DM1, particularly with large expansions in the congenital form and, experimentally, long (CUG)n-repeats interfere with myogenic differentiation. STR-1 is widely expressed and shares homology with Mindbomb, a ubiquitin-ligase in the Notch1-signalling pathway. The ubiquitin ligase ligand, Jagged2, controls STR-1 expression levels; when Notch1 is activated it translocates to the nucleus and stops differentiation by blocking muscle transcription factors. Hyperactivated Notch1 protein is increased in DM1 muscle and experimentally DM1 fibroblasts are able to transfer Notch1 abnormality to myogenesis in co-culture studies. When STR-1 is decreased Notch1 is increased and blocks differentiation, which can be reversed in DM1 cell culture by inactivation of mutant DMPK transcripts. This new pathway opens many possibilities to therapeutically interfere with any of the steps: for example STR- 1/(CUG)n interaction by competing molecules for binding, overexpression of STR-1, blocking gamma-secretase to inactivate Notch1, etc. 5. Gene expression profiling results in DM2 Ralf Krahe (USA). Extensive microarray studies using the Affymetrix GeneChip platform have been conducted during the last years comparing DM2 with DM1 and with myotonic myopathy patients negative for both DM1 and DM2 mutations (from hereon referred to as DMx patients) and two other muscular dystrophies (TMD titinopathy and dystrophinopathies). Using a 2-fold threshold for dysregulation, all three DM groups showed some genes differentially expressed relative to controls. Among these genes 614 were shared between the groups and 366 of the 614 were shared with the other dystrophies. Thus, 248 appear to be uniquely abnormally expressed in the DMgroup. DM1, DM2, and DMx patient biopsies show similar expression profiles, reflective of a common pathophysiology. Not surprisingly, many of the dysregulated genes are important in normal muscle physiology. Similar changes were seen in primary DM1 and DM2 myoblast cultures. Using primary DM1 myoblast cultures, some myoblasts differentiated while others showed a block or delayed differentiation. Expression profiles of DM1 myoblasts/myotubes that differentiated showed a profile more like that of normals than that of primary DM2 myoblasts. At later time points, however, DM1 myotubes showed profiles similar to those of wildtype early myoblasts. In DM2 cultures no differentiation problems were encountered. Some preliminary data using a new exon-tiling microarray platform (Affymetrix) were presented, which provided direct confirmation of aberrant splicing of titin (TTN), the DM-specific inclusion of exons in this gene. 6. Animal models for DM2 Laura Ranum (USA) described transgenic models they are developing to explore the RNA toxicity of CCUG expansion transcripts and the effects that expression pattern plays on the phenotypic similarities between DM1 and DM2. Transgenic lines expressing (CCUG) 300 and (CCUG) 5 transcripts have been generated and are currently being characterized. The conditional expression of this noncoding CCUG transcript will allow the evaluation of RNA toxicity, reversibility and variable expression. Initial phenotypic characterization of lines expressing the CCUG expansion is promising with several molecular changes observed in skeletal muscle. Ralf Krahe (USA) presented a DM2 mouse model, which is supposed to directly mimic the human situation by generating a humanized DM2 mouse, knocking-in (ki) a DM2 mutation [(TG)20(TCTG)11(CCTG)189] into the mouse homologous Znf9locus. The first examined 17 ki homozygotes are much smaller, and 39 heterozygotes are somewhat smaller than their littermates. Checking of other phenotype characteristics is underway. RT-PCR analyses have shown expression of the ki mutant allele in all tissues where Znf9 is normally expressed. A second model was presented, a transgenic mouse model similar to the one previously generated by Thornton and colleagues for DM1 [30], where a DM2 (TG)20(TCTG)11(CCTG)121 expansion has been inserted in intron 1 of a genomic expression vector of the human skeletal actin. Breedings of different founder lines are currently in progress. A third mouse model, which is essentially a ZNF9 overexpression model with a normal BAC (b814l21) clone containing the entire ZNF9 locus was also presented. The phenotype manifests a peculiar circulating behavior, heterozygous mice are smaller and show craniofacial abnormalities. 7. Myotonic dystrophy type 3 (DM3) and other non-dm1/dm2-families In 2004, a large French family with myotonic dystrophy phenotype combined with late FTD was published a possible DM3 family with suggestive linkage for a locus on Chr 15 [31]. However, in late 2005 Bjarne Udd was notified by the corresponding author of the paper that the

7 B. Udd et al. / Neuromuscular Disorders 16 (2006) family was not a DM3 candidate because linkage was incorrect and they had identified changes of Paget disease in the family and also identified the mutation in the VCP gene in the family [32]. This message was announced by Udd at the ICDM-5 conference in Quebec Ralf Krahe (USA) presented promising results on the mapping of a third locus for myotonic dystrophy on chromosome 16p. Myotonic dystrophy families (German, Spanish and Brazilian) lacking DM1 and DM2 mutations and excluded from linkage to chloride and sodium channel loci, have been studied by genetic linkage analysis. After reevaluation and exclusion of two German families (Benedikt Schoser and Frank Lehmannn-Horn, Germany), the current LOD-score is still above three with the remaining seven families in the analysis. All affected members in the families shared a region in 16p but on different haplotypes in the different families. More interesting, however, was the observation that non-dm1 and non-dm2 patients show identical abnormalities of CLCN1 splicing in the regions between exon 2 and 8, including the inclusion of intron 2, as seen in DM1 and DM2. Several other genes were also checked for DM-associated aberrant splice variants, including RYR1, MAPT, and TNNT2, all of which showed abnormal splicing in some or all patients. For further refinement of the genetic mapping, several pedigrees will be expanded. The search for the underlying mutation focuses on putative (CTG)-like repeat expansions, but proteins involved in RNA metabolism and splicing are also being considered as candidates. So far, 37 of 44 repeats in the linked region have been excluded. Josep Gamez (Spain) reported on Spanish families included in the DM3 linkage project. In the four families with suggestive linkage to Chr 16 there were 23 affected patients. Thirty percent of the patients had clinical myotonia, prominent myalgia with chronic back pain aggravated by exercise, no effect by pain treatment, no cognitive problem, depression in some because of pain, some with dysphagia, constipation. Proximal muscle weakness was only mild at 5- on MRC scale and muscle pathology consequently also with minor myopathic changes only and different from both DM1 and DM2. The range of onset was wide 9 53 years and usually with myalgia as the first symptom in the younger patients. In the first family with five affected: myalgia was present in 5/5, weakness in 1/5, and EMG myotonia in 4/5. One of these patients had first myalgia, then myotonia at age 26, cataracts at age 37, mild proximal weakness at age 47. CK was normal and calves were enlarged. In the second family onset varied between 20 and 37 years, myalgia in all, weakness in 2/5, myotonia 3/5, and cataracts at 37 in one patient. Muscle MRI at age 46 showed subfascial fatty degeneration, more pronounced in medial gastrocnemius. EMG was normal despite clinical fluctuation of myotonia. Besides, these four families under study for chr 16 linkage there are more families with myotonic dystrophy type disorder and unlinked to any of the known or suggestive loci. Frank Lehmann-Horn (Germany) described the phenotype of German families in the DM3 linkage project. In one family there was generalized EMG myotonia, proximal weakness, but also weakness of respiratory muscles and neck muscles, myalgia, no cataracts, cardiac arrhythmia, normal CK, and EMG myotonia also in 4/5 children of the proband. Onset of symptoms was at age 35 in the proband. In the second family, patients had painful cramps and had clinical myotonia by grip activation and percussion, myotonic pseudo-bursts were recorded on EMG, fasciculations occurred and patients had proximal weakness and some distal muscle atrophy. Muscle biopsy findings were classified as neurogenic. In a third family, unilateral atrophy prompted for genetic analysis of FSHD, which proved to be positive. On the other hand, clear myotonia and cataracts segregate in the same family members in a dominant fashion indicating that there are other genes involved besides FSHD. CK was normal. Benedikt Schoser (Germany) presented the muscle pathology, which showed features similar to DM2 with atrophic type 2 minifibers and both hypertrophic and smaller type1 fibers as well as increased amount of internalized nuclei. Christiane Schneider-Gold (Germany) presented a couple of German families not included in the DM3 linkage project and without DM1 or DM2 mutations. Patients had: myalgia, proximal weakness, cataracts, myotonia and high frequency discharges on EMG, thus qualifying for the definition of myotonic dystrophy. One patient in another family had: proximal weakness, EMG myotonia, mild cataracts, elevated CK. Giovanni Meola (Italy) also had seen patients with symptoms in the wide range of DMs but lacking the known mutations. One 54 years old sporadic woman had a history of fatigue for 6 years fatigue, multiple lipomas, ptosis, neck flexor and proxima limb weakness, no clinical myotonia, some EMG runs not typical myotonia, white matter lesions on brain MRI, ringfibers and some COX negative fibers in muscle pathology. Taken together findings suggested a mitochondrial background despite negative first genetic results. Bruno Eymard (France) gave an update of the French LeBer family with VCP mutated IBMPFD, and erroneously published as a possible DM3 family [31]. Indeed there was proximal muscle weakness, cataracts in 8/10, and EMG myotonia, besides frontotemporal dementia in the family. 7/10 patients had EMG myotonia, which is not previously well known in IBMPFD. In a previous series on IBMPFD in 42 patients, two patients were reported to have myotonia, but no other myotonic IBMPFD are known. Paget disease was very unremarkable in the family, but in retrospect the rimmed vacuolated myopathology was more pronounced than the occasional single vacuolated fibers seen in DM2. 8. Therapy and practical management Richard Moxley (USA) presented a comprehensive and careful overview on the current issues regarding therapeutic

8 410 B. Udd et al. / Neuromuscular Disorders 16 (2006) options and practical management of patients with DM2. The recommendations below in the Guidelines section are based on the presentation and the result of discussions during the presentation and accepted by the workshop. Benedikt Schoser (Germany) referred a recent retrospective study on the outcome of pregnancies and deliveries in 42 German DM2 women from 37 families. Results were very favorable: no case of congenital disorder occurred and no statistically significant higher frequency of complications was found in DM2 patients compared to an age matched control group, even if there was a tendency of low birth weight and more pre-term births in DM2 patients with onset of symptoms before the age of 30 years. Bjarne Udd (Finland) briefly presented messages coming from active patients and advocacy groups telling that the availability of DNA-tests should be increased to avoid the long latencies and difficult situations many DM2 patients have experienced because doctors and medical personnel were unfamiliar with DM2. Especially, easy access to updated general information on DM2 was requested. 9. Conclusions and measures for diagnostics and research Wolfram Kress (Germany) summarized discussions during the workshop to increase availability and accuracy of genetic DNA-tests in DM2 as the only reliable diagnostic definition. The workshop agreed on Guidelines for DNA Genetic Testing (see below) to ensure a lower threshold for requesting DNA tests. In addition, the represented European labs providing these DNA-tests agreed on regular sharing of samples with difficult results as a measure of continuous quality control. The system is already used between the three different French labs. Also the European ENOM group will be contacted for the same reasons. Charles Thornton (USA) summarized the prospects for successful scientific research on the exact molecular pathophysiology underlying DM2 and in parallel DM1. Tremendous advance during the last few years has brought research to the point of first in vitro tests of therapeutic approaches. The whole range of aberrantly spliced genes that are involved needs to be clarified as well as the protein content of the ribonuclear inclusions besides MBLNs. The weakness and progressive loss of muscle tissue is still without clear mechanism and clarifying the reason for type 2 fiber atrophy would be an important step in this direction. Completely new tools will be available during 2006 with the generation of a number of promising TG mouse models. The evaluation and assessment of phenotypic abnormalities should be standardized between the research groups in order to directly compare results in different models. New initiatives by the workshop-consortium: 1. One direct new clinical research project was initiated by the workshop: a retrospective study of the outcome of general anesthesia in DM2 patients to overcome the lack of reliable and confirmed data. The results will be used for more exact guidelines at the next workshop. The German group of scientists agreed to perform the study. 2. A web site for general information and Guidelines was initiated. The team in Rochester will have the main responsibility for the generation of an English website version and the workshop consortium will be responsible for the coordinated accessibility on the ENMC and MDA websites. The same information battery can be used and translated by Associations in other countries for their own use in other languages. Further initiatives for clinical research were discussed: to develop reliable clinical measures of myotonia trial of anti-myotonia therapy and questionnaire instruments to complement objective measurements. Definition on patterns of weakness over time and standardized clinical measures. Consider trial to evaluate efficacy of specific exercise programs. questionnaire instrument to describe and quantitate pain in its specific locations. trial of pain management using one or more of the treatments noted above. develop repository for lens tissues for research studies documentation of frequency of EKG and cardiac ultrasound abnormalities and heart attacks in DM2. Documentation of changes in CK and any toxicity during statin treatment. Standardized approaches to measure fatigue. Standardized measures of cognitive function and consider a trial of treatment. 10. Guidelines and recommendations Guidelines no. 1: indications for genetic DM2 testing Increasing numbers of patients without the full range of multi-organ symptoms associated with myotonic dystrophy have been verified with DM2 mutation. In the single patient, none of the common clinical key features: proximal weakness, myotonia, cataracts, elevated CK-values or established family history is absolutely mandatory for DM2 disease: the threshold for genetic testing of DM2 based on clinical findings should generally be lower than that usually required for DM1 genetic testing, especially if testing is started with the screening/exclusion test of the normal alleles at the DM2 locus.

9 B. Udd et al. / Neuromuscular Disorders 16 (2006) A very common clinical presentation is the combination of late adult onset of proximal weakness and EMG myotonia or just myotonia-like discharges. Other presentations may involve myalgia, various types of pain in limbs and trunk, as the most prominent symptom associated with one or more other feature of muscle disease. Findings typical for DM2 on muscle biopsy may be another indication for genetic testing. A considerable number of patients with verified DM2 mutation have had very unusual phenotypes requesting an open mind by the clinician regarding DM2 disease. Considering that the risk of fatal events later in life for a child born with the DM2 mutation is low, genetic testing in childhood can be considered only in certain families with established exceptionally prominent cardiac involvement Guidelines no. 2: recommendations for management Clinical myotonia is rarely severe or disabling. However, chronic back stiffness, troublesome grip myotonia, and frequent thigh muscle strain and pain should prompt consideration of a trial of mexiletine. Baseline blood count, electrolytes, and liver function tests (including gamma glutamyl transferase level) are recommended along with EKG. Mexiletine 150 mg twice or three times daily with food is often helpful. If mexiletine is beneficial, trials at higher dosage may be appropriate to achieve optimal response. Serial monitoring of blood tests and EKG are recommended. Mexiletine may also have a beneficial effect in controlling the frequent and difficult to treat muscle pain mentioned below. The workshop group recommends serial monitoring of muscle weakness with function testing and suggests use of supervised aquatic therapy and land based resistive training to ameliorate fatigue and to maintain strength. Different remedies have been tried for muscle pain in DM2 without consistent benefit from any specific medication regimen. Anecdotal experience has suggested that in addition to mexiletine that some patients obtain relief from carbamazepine and anti-spasticity medication (if hyperreflexia is present: addition of baclofen, tizanidine, or gabapentin may prove helpful). Referral to a pain specialist may be necessary if completely disabling pain persists. Cataracts are treated by surgical removal as necessary. Cardiac fatalities have occurred. At present the risk for such events is not clear, but it is likely to be low. However, the workshop group recommends serial monitoring of the EKG similar to that for DM1 at 6 12 month intervals and prompt referral for cardiology consultation if EKG abnormalities or clinical signs suggestive of cardiac dysfunction occur. Current medications may need re-evaluation regarding risk/benefit if arrhythmia appears. While manifest cardiomyopathy is rare, coronary heart disease in DM2 patients is not. Management of cardiac problems needs to be co-ordinated with the primary care physician and the cardiologist. Atherosclerosis and related hyperlipidemia are frequent in DM2 and seem to correlate closely with the insulin resistance that develops in patients. Serial monitoring of the lipid profile and intermittent assessment of oral glucose tolerance testing (including measurement of serum insulin values at 0, 30, 60, 120, 150, and 180 min timepoints after glucose ingestion). Statin treatment is often necessary to control the hyperlipidemia in patients with DM2 and can pose a most challenging problem. Patients with DM2 typically have myalgias and elevations of serum CK in the absence of statin treatment. Once statin treatment has begun, it is difficult in certain DM2 patients to determine if they are developing a statin related myopathy. Serial measurements of creatine kinase and careful documentation of the bouts of muscle pain are helpful in establishing the usual fluctuations in these manifestations of DM2, and such measurements can aid the clinician in deciding if statin toxicity has developed. Elevated creatine kinase levels can be aggravated by exercise or muscle trauma and it is desirable to repeat the measurement after several days of inactivity. Unfortunately, there is no specific test available to distinguish statin myopathy from the clinical manifestations that can occur in DM2. Not all DM2 patients have pronounced or severe adverse reactions to statins, but if they develop, discontinuing treatment indefinitely may be the most prudent course of action. Moderate elevations of total and LDL cholesterol in older patients may be less harmful than the toxic myopathy that can occur. In any case, statin treatment needs coordination with primary care physician and cardiologist. Other treatment concerns need coordination with primary care physician and other care providers. These include treatment of hypothyroidism and gonadal failure (impotence) with thyroid and testosterone replacement. Risks for complications in general anesthesia are well known in DM1 and the workshop group recommends for the present that the established guidelines for DM1 be followed for patients with DM2. No definitely increased risk of anesthesia has yet been reported in individuals with DM2. However, until there is a more detailed understanding of the clinical spectrum of disease in DM2, it is useful and prudent to adhere to the guidelines established for DM1. In any case, careful pre-operative review of the medications each patient is receiving and discussion with the anesthesiologist and surgeon concerning complications of holding or delaying the reinitiation of one or more of these medications is needed. An elevation of serum potassium can aggravate muscle stiffness and pain in some patients with DM2. It is desirable to keep serum potassium levels at w4 mequiv./liter, especially in the immediate post-operative period.