111th ENMC International Workshop on Multi-minicore Disease. 2nd International MmD Workshop, 9–11 November 2002, Naarden, The Netherlands

Neuromuscular Disorders 14 (2004) 754–766 www.elsevier.com/locate/nmd

Workshop report

111th ENMC International Workshop on Multi-minicore Disease. 2nd International MmD Workshop, 9–11 November 2002, Naarden, The Netherlands Heinz Jungblutha,b, Alan Beggsc, Carsten Bo¨nnemannd,e, Kate Bushbyf, Chantal Ceuterick-de Grooteg, Brigitte Estournet-Mathiaudh, Nathalie Goemansi, Pascale Guicheneyj, Alain Lescurek, Joe¨l Lunardil, Francesco Muntonia, Ros Quinlivanm, Caroline Sewrym, Volker Straubn, Susan Treveso, Ana Ferreiro j,* a

Dubowitz Neuromuscular Centre, Imperial College, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK b Department of Paediatric Neurology, Guy’s Hospital, St Thomas Street, London, UK c Genetics Division, Harvard Medical School, Boston, MA, USA d Georg August Universitat Go¨ttingen, Kinderklinik and Poliklinik, Go¨ttingen, Germany e The Children’s Hospital of Philadelphia, Philadelphia, PA, USA f Institute of Human Genetics, International Centre for Life, University of Newcastle upon Thyne, Newcastle upon Thyne, UK g Born Bunge Foundation, Department of Neuropathology, University of Antwerpen, Wilrijk, Antwerpen, Belgium h Hoˆpital R. Poincare´, Sce. de Pe´diatrie, Re´animation Infantile et Re´e´ducation Neuro-respiratoire, Garches, France i Department of Pediatrics, University Hospital Gasthuisberg, K. U. Leuven, Leuven, Belgium j INSERM U582/Institut de Myologie, Groupe Hospitalier Pitie´-Salpeˆtrie`re, Baˆtiment Babinski, 47 Boulevard de l’Hoˆpital, 75013 Paris, France k UPR 9002 du CNRS, IBMC, Strasbourg, France l Laboratoire de Biochimie de l’ADN, CHU de Grenoble, France m Robert Jones and Agnes Hunt Hospital NHS Trust, Oswestry, UK n Department of Pediatrics, University of Essen, Essen, Germany o Department of Anesthesia and Research, Kantonsspital Basel, Basel, Switzerland Received 9 June 2004; received in revised form 7 July 2004; accepted 7 July 2004

1. Introduction. MmD overview Fifteen clinicians and basic scientists from six countries convened on 8th and 9th November 2002 in Naarden, The Netherlands for the 2nd ENMC sponsored Workshop on Multi-minicore Disease (MmD). The 1st ENMC sponsored workshop on MmD in May 2000 had lead to collaborations between the participating groups, resulting over the ensuing 2 years in a rapid advance in the understanding of this autosomal recessively inherited congenital myopathy. The main objective of this workshop was to discuss the recent identification of two genes (RYR1 and SEPN1) involved in at least two of the four MmD phenotypes that had been defined during the previous MmD consortium meeting.

* Corresponding author. Tel.: C33 1 42165704; fax: C33 1 42165700. E-mail address: [email protected] (A. Ferreiro). 0960-8966/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2004.07.007

In the light of these data, the clinical and morphological experience of the different groups was shared and discussed in order to further define the diagnostic criteria for MmD and its phenotypical spectrum, and to establish phenotype– genotype correlations. A further focus of the meeting was to analyse those groups of patients with untypical features and as yet unresolved genetic basis. After the welcoming address by J. Andoni Urtizberea, Research Director of the ENMC, the workshop was introduced by Ana Ferreiro and Heinz Jungbluth, who gave an overview of MmD. MmD is a congenital myopathy morphologically defined by localized areas of mitochondrial depletion and sarcomere disorganization, running to a limited extent along the longitudinal muscle fibre axis (‘minicores’) and present in both type 1 and type 2 fibres. From a clinical point of view, MmD is an early-onset, heterogeneous condition, in which four different subgroups have been distinguished [1–3]. The phenotype of the most

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instantly recognizable, ‘classical’ MmD form is characterized by the axial predominance of muscle weakness, severe scoliosis and respiratory insufficiency. The moderate form consists of generalized muscle weakness pronounced in the pelvic girdle and often prominent hand involvement with weakness, amyotrophy and hyperlaxity; scoliosis and respiratory involvement are mild or absent in this group of patients. The two other forms of the disease are characterized by a clinical picture similar to the classical one but including ophthalmoplegia, and by antenatal onset with arthrogryposis, respectively. None of the forms of MmD is associated with significantly increased serum creatine kinase levels, intellectual or primary cardiac involvement. No episode of malignant hyperthermia or family history of anaesthetic deaths have thus far been retrieved in any of the MmD families.

2. MmD and RYR1 Excitation–contraction (EC) coupling in skeletal muscle involves mechanical and functional interactions between Ca2C release channels (dihydropyridine receptor and ryanodine receptor) and modulating proteins (triadin, calsequestrin, calmodulin, FKBP12). The ryanodine receptor type 1 gene (RYR1, chromosome 19q13.1) encodes a calcium release channel (RyR1) localized at the skeletal muscle sarcoplasmic reticulum, where it plays a key role in EC coupling and calcium homeostasis of the skeletal muscle in mammals. Since in the late 1990s, heterozygous RYR1 mutations were known to cause two related but different autosomal dominant conditions: malignant hyperthermia (MH), a pharmacogenetic disorder of skeletal muscle Ca2C regulation triggered in genetically predisposed individuals by exposure to common anesthetic agents [4], and the congenital myopathy central core disease (CCD). CCD [MIM 117000] is characterized by core lesions that have sharply defined borders, involve exclusively type 1 fibres and are considered to extend throughout the entire fibre length; they are more often central and unique, but can be predominantly eccentric [5] or multiple within one fibre. The differences in core length, clinical phenotype and transmission pattern between MmD and CCD have generally led to consider them as separate entities. However, recent studies have shown that homozygous mutations of RYR1 are responsible for at least two of the MmD phenotypes. The first session of the workshop was devoted to the presentation of these studies by their respective authors, and to discussion of the nosological implications of these unexpected molecular findings. 2.1. The moderate form of MmD with hand involvement: genetic basis and phenotype re-evaluation Ana Ferreiro summarised the studies concerning this form of MmD, initially described in three sisters born from

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consanguineous Algerian parents [3]. Their clinical picture was characterized by early-onset, moderate weakness predominant in pelvic girdle, axial muscles and hands, together with joint hyperlaxity. A genome-wide screening performed on the series of MmD patients from the Institut de Myologie (Paris) led to map the disease to chromosome 19q13.1 in this Algerian family and, subsequently, in three additional families with a similar phenotype. This locus was excluded in 16 other MmD families with a ‘classical’ phenotype. Unexpectedly, the recessively linked 3 cM-region contained the gene RYR1, previously implicated only in dominant conditions (CCD, MHS). In the Algerian family, analysis of the entire RYR1 cDNA, done in collaboration with J. Lunardi’s group (Grenoble), disclosed a novel homozygous missense mutation (P3527S) in exon 71 [6]. In the three remaining families, screening of exon 71 and of exons 94–106 (encoding the C-terminal domain of RyR1) using genomic DNA did not show any mutation. A phenotype re-evaluation of the four families linked to 19q13.1 confirmed that all parents were clinically normal, and that the eight affected cases shared a common phenotype [6]. All showed infancy or childhood onset, moderate muscle weakness (generally 4–5—according to the MRC scale) involving mainly the pelvic girdle, neck and trunk flexors and hands; hand hypotonia, hyperlaxity and moderate weakness were associated with intrinsic hand muscle amyotrophy. Moderate hyperlaxity was also present in the remaining limb joints; four patients had mild or moderate heel contractures. Only one case showed minimal, stable scoliosis. Although three cases had a moderate reduction of vital capacity (52–70% of the predicted values), none developed respiratory failure. Radiological examination of lower limb muscles in four patients, using CT scan or MRI, showed also a constant pattern, with preferential involvement of gluteus maximus and quadriceps, although the rectus femoris was relatively spared. Actually, most of the clinical and radiological findings in this group of patients are consistent with the classically described CCD features, with the exception of joint and particularly hand hyperlaxity that might have been underestimated in CCD [7]. Despite the clinical homogeneity, the severity of the disease was variable. The two most severe cases presented with congenital club feet, neonatal hypotonia and delayed motor development; the eldest one, now aged 25 years, can hardly walk with help. Muscle weakness remained stable in most cases. In the Algerian family with the P3527S mutation, two biopsies performed in the two eldest sisters at 2 (left deltoid) and 4 (right quadriceps) years of age, respectively, showed type 1 fibre uniformity, associated with multiple minicores in the second case. In view of the genetic results, we performed additional muscle biopsies in the three patients at adulthood. A right deltoid sample from the eldest sister showed a dramatic histopathologic change with extensive

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fibre loss and sharply defined, long core lesions in the few spared fibres. The new samples from her two sisters disclosed a pattern typical of central core disease with rods. MH sensibility was excluded after an in vitro contracture test. Deltoid biopsies from both asymptomatic parents showed no core lesions. In conclusion, this subgroup of families linked to 19q13 represent the first variant of CCD with genetically proven recessive inheritance and transient presentation as MmD. This work established the genetic heterogeneity of MmD and demonstrated that classical morphological lesions may undergo age-related modifications and therefore represent necessary but unreliable diagnostic criteria in the complex overlap of phenotypes between MmD and CCD. 2.2. The ‘CCD-like’ phenotype of MmD: clinical and genetic data Heinz Jungbluth presented clinical and genetic findings in the group of patients with the moderate form of MmD with predominant hip girdle involvement and sparing of respiratory muscles. Thirteen out of 34 patients from the United Kingdom MmD cohort confirmed to this particular phenotype; five of them currently still under follow-up. Despite distinct histopathological findings, marked clinical overlap between this particular phenotype and ‘classical’ central core disease (CCD) was noted. Clinical features were characterized by late onset in childhood with static or only slowly progressive course, predominant hip girdle weakness with absence of significant bulbar or respiratory involvement, myalgia related to exercise and cryptorchidism in more than two-thirds of affected males. Ligamentous laxity was commonly associated and pronounced in the distal upper limb. One boy whose brother was only mildly affected presented with bilateral congenital dislocation of the hips and arthrogryposis. Muscle MRI of the thigh and leg muscles performed in three patients showed a characteristic and consistent pattern of selective muscle involvement identical to the pattern observed in individuals with CCD secondary to dominant mutations in the RYR1 gene [8,9]. Haplotyping studies in two informative families with three affected individuals were compatible with linkage to the RYR1 locus on chromosome 19q13.1. Subsequent mutational screening of the RYR1 gene focused on exons 39 and 45 previously associated with a congenital myopathy phenotype, and C-terminal exons 98–103 recently identified as a mutational hotspot in CCD [10]. A homozygous G14545A change in RYR1 exon 101 resulting in a V4849I substitution was identified in the first family, whereas no mutation could be identified in the second family [8]. Mutational analysis of additional RYR1 exons is currently in progress in this family. These findings provided the genetic basis for the observed clinical and radiological overlap between CCD and MmD with predominant hip girdle involvement, and confirmed that recessive mutations in the RYR1 gene may

account for a subset of patients with a histopathological diagnosis of MmD. These observations demonstrated that histopathological changes associated with recessive RYR1 mutations are variable and may comprise both central cores and minicores, and that muscle MR imaging may be a useful ancillary tool to indicate RYR1 involvement in cases with equivocal histopathology. 2.3. RYR1 mutation analysis and recessive core myopathies The RYR1 gene located on chromosome 19q13.1–13.2 has been involved in several congenital myopathies, including central core disease (CCD) [5,11–14] and malignant hyperthermia, a sub-clinical myopathy occasionally associated with histological core pictures [15]. Although considered as genetically heterogeneous, autosomal dominant CCD was linked to the RYR1 gene in a majority of investigated families. Most of the mutations identified so far in the RYR1 gene mapped to the C-terminal domain of the protein [5,11–14]. Joel Lunardi summarised the experience of his group on RYR1 mutation analysis, particularly focusing on recessive core myopathies. Their contribution to the genetic investigation of the four homogeneous, autosomal recessive MmD families described above provided evidence that the disease in these families is associated with the RYR1 gene. Phenotype re-evaluation showed that the pathological condition may actually represent a variant form of CCD with autosomalrecessive inheritance and transitory presentation as MmD. The RYR1 gene is a large and complex gene comprising 106 exons that span over 150.000 base pairs. Because of the large size of the gene, J. Lunardi’s group has established a particular strategy for mutation analysis. Full mutation screening is usually only performed on cDNA obtained from a skeletal muscle biopsy; the 31 overlapping amplified fragments that represent the whole RYR1 cDNA are subsequently sequenced. Alternatively, in absence of a muscle biopsy, RYR1 mutation screening is performed on genomic DNA and restricted to the 3 0 end of the gene (exons 94–106), encoding the transmembrane, channel-forming domain that contains most of the mutations associated with CCD. In the three affected sisters of a consanguineous family, this strategy disclosed a single homozygous amino acid change, P3527S, resulting from a C to T transition at position 10579 of the cDNA [6]. The mutation was present at the heterozygous level in the two unaffected parents. The change affected a very well-conserved proline residue in a region of RyR1, where several putative calmodulin binding sites have been assigned. The recessively expressed P3527S mutation was identified in a domain very different either from the MHS1 and 2 domains (where most mutations associated with a MH phenotype are clustered) or from the CCD-associated C-terminal domain. The recessive expression of P3527S may be related to this particular localization. Indeed, its position in exon 71, where no other

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mutations have been described, allows to hypothesize that this mutation does not affect either the interaction with the DHPR or the channel function. Therefore, P3527S would have minor functional effects unless both alleles are affected. Involvement of the RYR1 gene in autosomal recessive core myopathies was also suggested in another family, studied by Francesco Muntoni’s group, where a homozygous V4849I RYR1 mutation was identified in the propositus, presenting with a recessive core myopathy [8]. Both parents were clinically unaffected carriers. Interestingly, the same heterozygous V4849I mutation had been previously identified in a dominant Australian malignant hyperthermia pedigree without other neuromuscular symptoms (Davis, personal communication), suggesting that homozygosity for this mutation is required for the full clinical expression of the congenital myopathy phenotype. The mutation mapped to the C-terminal hot spot domain for CCD mutations, clearly indicating that functional analysis of the different mutations and identification of additional mutations of the RYR1 gene are needed for a better understanding of the pathophysiological processes associated with CCD and MmD.

gene decreases the EC50 of 4-chloro-m-chresol induced calcium release approximately two-fold [16]. They also tested the functional characteristics of EBVtransformed lymphocytes from patients affected by CCD carrying different mutations in the C-terminal, transmembrane domain of the skeletal muscle RyR1. By studying intracellular calcium homeostasis, they found that cells carrying the endogenous, heterozygous RYR1 mutations Arg4893Trp, Arg4861His, Ile4898Thr, Gly4899Arg and the D4863–4869 deletion, all had significantly smaller intracellular Ca2C stores than cells carrying a wild type RyR1 or carrying the MH-linked mutation Val2168Met. They also found that the cell lines established from CCD patients exhibited release of calcium from intracellular stores in the absence of pharmacological activators of the RyR1 receptor. Such unprompted calcium transients were never seen in cell lines of control individuals or individuals carrying the MH-linked mutation Val2168Met, and could be blocked by the addition of dantrolene, an inhibitor of RyR1. Furthermore, dantrolene pre-treatment restored the thapsigargininduced Ca2C-peak to control levels supporting the view that the smaller intracellular calcium stores observed in cells from patients carrying mutations in the RYR1 C-terminal domain are due to a leaky RyR1 calcium channel.

2.4. Functional study of RYR1 mutations using lymphoblastoid cell lines

2.5. MR imaging studies in RYR1-related myopathies

Susan Treves (Basel, Switzerland) presented the model developed by her group to study the functional properties of mutated RyR1. Central core disease (CCD) and malignant hyperthermia (MH) result from a defect in the components involved in the mechanisms underlying skeletal muscle excitation–contraction coupling. So far, the study of the functional properties of ryanodine receptor (RyR1) channels carrying point mutations associated with MH and CCD has relied mostly on the use of heterologous cells overexpressing the RyR1. However, human B-lymphocytes were recently shown to also express the skeletal muscle isoform of the RyR calcium channel. Therefore, this cell type offers a good model to study the RyR1 channel in native conditions. In addition, the ‘ectopic’ RyR1 expression by this cell type may imply the involvement of the immune system in some aspects of the pathophysiology of MH and CCD. S. Treves’ group carried out a set of experiments to unequivocally demonstrate the expression of type 1 RyR in EBV immortalised B-cell lines established from control individuals and MH susceptible (MHS) individuals carrying the RyR1 V2168M mutation. They also investigated the functional effects of ‘natural’ mutations in the RYR1 gene in these EBV-immortalised B lymphocytes by analysing their sensitivity to the RyR type 1 specific agonist 4-chloro-mcresol. Their results show that EBV immortalised lymphocytes from normal donors are sensitive to 4-chloro-m-cresol, and that the presence of the V2168M mutation in the RYR1

Heinz Jungbluth presented muscle MR imaging findings in congenital myopathies secondary to mutations in the RYR1 gene [9]. Muscle MRI has been suggested as a useful ancillary tool to inform the choice of confirmatory genetic tests in some forms of the muscular dystrophies [17], but systematic MRI studies correlating genetic and imaging data are currently not available for the congenital myopathies. Eleven patients from eight families with mutations in the RYR1 gene (heterozygous nZ6, homozygous nZ1) or confirmed linkage to the RYR1 locus (nZ1) had MRI scanning of their lower limb muscles. All mutations identified exclusively concerned exons 101–102 affecting the C-terminal portion of the protein, a recently identified hotspot for mutations associated with a congenital myopathy phenotype [10]. Eight patients had a muscle biopsy and in three others the diagnosis of a congenital myopathy was established because of suggestive histopathological features in a similarly affected sibling. Four patients had the typical histopathological appearance of central core disease. In the remaining cases histopathological changes were either non-specifically myopathy (nZ1) or showed additional features such as marked increase in fatty and connective tissue (nZ2) or prominent minicores and nemaline rods (nZ1). All patients showed a consistent pattern of selective muscle involvement. This consisted of (a) within the thigh: selective involvement of vasti, sartorius, adductor magnus and relative sparing of rectus, gracilis and adductor longus;

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(b) within the lower leg: selective involvement of soleus, gastrocnemii and peroneal group and relative sparing of the tibialis anterior. The degree of abnormal signal intensities correlated with the clinical severity in individual patients and the histopathological changes in specific muscles. The pattern of selected muscle involvement was distinct from imaging findings reported in other neuromuscular conditions including SEPN1-related myopathies and nemaline myopathy. These results suggest that patients with congenital myopathies secondary to C-terminal RYR1 mutations have a specific pattern of muscle involvement irrespective of the variability of histopathological changes. Muscle MRI may therefore supplement clinical assessment and aid selection of genetic tests particularly in patients with non-diagnostic or equivocal histopathological features. Histopathological changes may vary greatly even in the same patient depending on the selected muscle biopsy site. 2.6. Clinical re-evaluation of other RYR1-related patients and similar but genetically unresolved cases Various clinicians summarized clinical findings of patients with MmD under review at different centres. This comprised patients with confirmed or suspected involvement of the RYR1 gene, and other clinically similar cases whose genetic basis remains unsolved. Ros Quinlivan (Oswestry, UK) presented clinical features from 10 children with minicores on muscle biopsy and distinguished four different categories characterized by the following features: (1) distal arthrogryposis, facial, axial and hip girdle weakness in three cases, one isolated case and two siblings from a consanguineous family with finger flexion contractures in the mother and maternal aunt; (2) ophthalmoplegia, ptosis, generalised muscle atrophy and scoliosis in a 3-year-old boy with similarly affected mother and maternal aunt; (3) severe exercise intolerance associated with muscle pain in children with mild hip extensor weakness and hamstring contractures on examination (nZ3), following a presentation with neonatal hypotonia and feeding difficulties or normal early development; (4) early-onset scoliosis, neck retraction and respiratory impairment (nZ3) with additional cardiomyopathy in one 19-year-old female. Ptosis was a common additional finding in the first three categories, and mild learning difficulties were observed in few children. Autosomaldominant inheritance was suggested in the family with external ophthalmoplegia, whereas other cases were sporadic or compatible with recessive inheritance. Screening of RYR1 C-terminal exons 98–103 has already been performed or is currently underway in these families, but no mutations have been identified so far. One additional autosomaldominant family with two affected children and an affected mother was found to have a heterozygous missense mutation in RYR1 exon 101, resulting in a Arg 4893Trp amino acid substitution. Muscle biopsies in this family

showed central cores in the mother, but minicores in the two affected children, emphasizing the wide histopathological spectrum associated with mutations in the RYR1 gene. Volker Straub (Essen, Germany) gave further details of clinical findings in one of the families with moderate MmD linked to 19q13.1 and genetically studied in Paris. Two affected boys born to non-consanguineous German parents showed a similar clinical picture with different degrees of severity. The more severely affected brother presented at birth with bilateral congenital foot deformities, and showed delayed motor development and delayed growth and weight gain. Muscular atrophy and weakness was more pronounced in his limb muscles compared to his trunk muscles. Distal muscles were more severely affected than proximal muscles. At the age of 2 years the patient presented with flexion contractures in both wrists and ankles, weak muscle tendon reflexes, and unilateral ptosis. At the age of 6 and 7 years achillotenotomy was performed. At this age, growth retardation became even more obvious. The disease showed slow progression until the present age of 10 years. The older affected brother presented with much milder symptoms. His major clinical signs at the age of 13 years are episodes of myalgia, generalized muscle weakness with normal muscle bulk and weak tendon reflexes. Both brothers had normal levels of serum creatine kinase, pathological EMGs, and large but short multi-minicore lesions in the muscle biopsy taken from the more severely affected case at the age of 5 years. V. Straub presented also the clinical findings in another child born of non-consanguineous German parents whose phenotype was marked by prominent distal muscular weakness and hypotonia, with the limbs more affected than the trunk and the hands and fingers more affected than the legs and toes. She had prominent facial weakness, and developed contractures of the elbows, drop hands, hyperlordosis, toe walking and slight growth retardation about the age of 12 years. Her serum CK levels were slightly elevated. She showed normal nerve conduction velocities and slight changes in the EMG and EEG. At the age of 19 years, she had still marked facial weakness and severe weakness and atrophy of the lower arms. A muscle biopsy showing dystrophic changes was taken from the extensor digitorum muscle at the age of 5 years. A muscle biopsy from the femoral quadriceps muscle at the same age revealed no dystrophic changes, but characteristic signs of multiminicore disease. The genetic basis in this case remains unknown. All children had a normal intellectual development and none showed signs of cardiac involvement and so far no signs of malignant hyperthermia. 2.7. MmD with ophthalmoplegia Heinz Jungbluth presented clinical and genetic findings of eight patients from three families with features of MmD and additional external ophthalmoplegia [18]. Clinical

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features in this group of patients were characterized by onset in the first year of life (nZ8/8), feeding difficulties and/or weight below the third centile (nZ3/8), prominent axial and shoulder girdle involvement (nZ6/8) and moderate scoliosis (nZ3/8). The clinical phenotype of these patients was more reminiscent of the ‘classical’ phenotype of MmD rather than the form resembling CCD. Although none of these patients required nocturnal ventilation, in most cases forced vital capacity was moderately reduced and one patient suffered a life-threatening episode requiring intermittent ventilation following an intercurrent respiratory illness. Muscle MRI of the thigh and leg muscles was performed in four patients. Muscle MRI findings in patients with MmD and external ophthalmoplegia have not been previously reported and showed some similarities with muscle MRI findings in patients with dominantly inherited CCD [19], namely sparing of gracilis compared to sartorius and gastrocnemii compared to soleus. However, other muscle groups were more diffusely involved and the degree of overall muscle wasting was more pronounced. The observed pattern was, however, distinct from MR imaging findings in patients with MmD carrying SEPN1 mutations, where gastrocnemii are typically more affected than the soleus [17]. Muscle MRI may therefore contribute to differentiate patients with MmD and external ophthalmoplegia secondary to recessive RYR1 mutations from patients with other disorders. Haplotyping studies in three informative families with eight affected individuals were compatible with linkage to the RYR1 locus on chromosome 19q13.1. Screening of selected RYR1 exons 39 and 45, and C-terminal exons 98–103 revealed a heterozygous T7268A base change in RYR1 exon 45 in one family with four affected and one unaffected sibling. The resulting Met2423Lys amino acid substitution was identified in all affected individuals, but also the unaffected father, suggesting compound heterozygosity and the presence of an as yet unidentified allelic mutation. No mutation in the selected RYR1 exons could be identified in other families. These findings suggested that the form of MmD with external ophthalmoplegia is also associated with recessive mutations in the RYR1 gene. Clinical discordance between CCD and MmD and external ophthalmoplegia may be explained by mutations affecting functionally distinct domains of the RyR1 protein. The Met2423Lys affects a RyR1 domain previously predominantly associated with a malignant hyperthermia phenotype, and a MH causing mutation has been recently identified in close proximity [20]. These findings may indicate a continuum between congenital myopathy and MH phenotypes associated with RYR1 mutations. Further evidence of association between RYR1 mutations and partial external ophthalmoplegia was provided by Ana Ferreiro. One of the Algerian patients having a moderate form of MmD due to a homozygous RYR1 mutation

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(P3527S) first complained of occasional diplopia associated with some particular head positions at the age of 25 years. Clinical examination at that moment disclosed a clear, although, moderate limitation of upward gaze, particularly on abduction, while primary, lateral and downward gaze were normal. A brain MRI showed no intercurrent abnormalities. External eye movements had been repeatedly examined in the previous 3 years and found to be normal. The observed partial ophthalmoplegia remains stable after 1 year of follow-up. Therefore, ophthalmoplegia in cases with RYR1 mutations maybe subtle and appear as a late sign, even in adult patients showing an otherwise stable course of the disease.

3. MmD and SEPN1 3.1. SEPN-related myopathy. From phenotype to genetics and back Ana Ferreiro summarized the studies that led to the genetic characterization of classical MmD [21]. Based on clinical and morphological data, they suspected a relationship between classical MmD and the selenoprotein N gene, SEPN1, located on chromosome 1p36 (RSMD1 locus) and responsible for congenital muscular dystrophy with Rigid Spine Syndrome (RSMD) [22]. A genome-wide screening followed by the analysis of 1p36 microsatellite markers in 27 informative MmD families demonstrated linkage to RSMD1 in eight families. All showed an axial myopathy with scoliosis and respiratory failure, consistent with the most severe end of the ‘classical’ MmD spectrum. Linkage to RSMD1 was excluded in 19 MmD families, including nine classical ones, demonstrating the genetic heterogeneity of classical MmD. Screening of SEPN1 in the eight linked families and in 14 classical sporadic cases disclosed nine mutations affecting 17 patients (12 families); six were novel mutations; three had been described in RSMD patients. These unexpected molecular findings prompted a clinical re-evaluation of all the patients with SEPN1 mutations. All showed homogenous features, marked by congenital and severe axial weakness with early scoliosis and respiratory failure. Failure to acquire head control was a constant early sign. In contrast, 13 cases were able to walk at a normal age; for this reason, the diagnosis of myopathy was not evoked in some cases until late teens. Muscle weakness and slenderness were always more marked in axial groups, particularly in neck flexors (0–2—MRC); none of these patients has ever been able to lift their head from the supine position. In contrast, limb muscles were relatively preserved (3–4) and quadriceps power was often normal. Distal amyotrophy of the inner thighs (‘bracket-like thighs’) and calf amyotrophy were constant and characteristic. Most patients had a nasal, high-pitched voice, and a variable degree of facial weakness. Cervical contractures were not early findings; in fact, some patients’ head dropped forwards throughout infancy

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and childhood. In most patients (13) but not all, a variable degree of spinal rigidity developed later. Most young patients showed moderate distal hyperextensibility; approximately half of them developed late, mild to moderate knee and elbow retractions. Scoliosis, present from the mean age of 8.5 years, was generally cervicodorsal, associated with dorsal lordosis and a lateral trunk deviation, and steadily progressive. Arthrodesis was required in nine cases and resulted in orthopaedic and respiratory improvement. Restrictive respiratory insufficiency required early respiratory assistance in all but the two youngest patients now aged 10 and 5 years. Six patients were tracheotomized. There was a clear diaphragm involvement but, in addition, nocturnal desaturations and frequent apnea periods were disclosed by polysomnographic studies, even at early stages of the disease with fairly preserved vital capacity. Failure to thrive was present in all but one case. There was no heart or mental involvement. Serum CK levels were never more than four times the upper normal value. Muscle weakness remained stable in 13 cases (76%) and was slowly progressive in four. Most adult patients remain able to walk; only the most severe case lost gait at 8 years of age. Analysis of three deltoid biopsies from typical RSMD cases revealed a wide myopathological variability, ranging from a dystrophic to a congenital myopathy pattern. A variable proportion of minicores was found in all the samples. In conclusion, the clinical, morphological and molecular overlap between the RSMD1 and the classical form of MmD with SEPN1 mutations suggest that they are best understood as a single entity, for which the term of SEPN-related myopathy is proposed. This work demonstrates also the genetic heterogeneity of classical MmD and implicates reconsidering the nosological boundaries between MmD and RSMD. 3.2. Selenoproteins and the SEPN1 gene. Mutation update and preliminary data on protein subcellular localisation Pascale Guicheney presented additional details on selenoproteins and SEPN1 mutations. Selenoproteins contain at least one selenocysteine, which is the major biological form of selenium in bacteria, plants and animals. Selenocysteine is the 21st amino acid, whose chemical structure is the same as a cysteine with a selenium atom instead of sulphur, and it is co-translationally incorporated in the active sites of selenoproteins. Nineteen selenoproteins have been identified in humans corresponding either to families of proteins such as glutathione peroxidases, thioredoxin reductases and iodothyronine deiodinases, or to unique enzymes such as the selenophosphate synthetase. All selenoproteins with known functions are enzymes, the selenol group, which is ionized at physiological pH, confering more reactivity to these proteins than a thiol group.

All selenoproteins share some characteristics at the molecular level directly linked to the incorporation of the selenocysteine into the protein. They present (i) an in-frame UGA codon in the coding sequence, which instead of being read as a termination codon is recognized as a selenocystein codon, (ii) a specific sequence with a stem-loop structure called SECIS (SElenoCysteine Insertion Sequence) in the 3 0 untranslated region of the mRNA. The SECIS structure contains a conserved quartet of non-Watson–Crick-interacting nucleotides, and an apical loop containing a nonpaired AA motif. The secondary structure of the SECIS element is crucial for the recognition of UGA as a selenocysteine codon. Selenoprotein N (SEPN) is a protein expressed at the transcript level in numerous tissues including skeletal muscle. Mutations in the SEPN1 gene cause an autosomal recessive myopathy both in subjects initially diagnosed as RSMD (Rigid Spine Muscular Dystrophy) and in classical MmD patients. So far, SEPN1 mutations have been identified in 48 patients; 28 were carriers of a homozygous mutation, while 20 had compound heterozygous ones. Of the 29 different mutations identified, two-thirds induced a premature stop codon (nonsense, splicing mutations, insertions or deletions). Ten missense mutations occurred in three regions of the protein (i) in a conserved domain probably corresponding to a functionally important domain of the protein, (ii) in or in the vicinity of the UGA codon, (iii) in the SECIS sequence. Mutations of the selenocysteine UGA codon or the SECIS element probably prevent the incorporation of the selenocysteine and induce the formation of a truncated or an abnormal protein. Several founder mutations have been identified in European populations, explaining the occurrence of rather frequent homozygous mutations in non-inbred families. SEPN function is unknown. P. Guicheney’s group developed antibodies to better characterize the protein, and showed that, despite a previous detection of two SEPN1 transcripts, the main SEPN1 gene product corresponds to a 70 kDa protein, containing a single selenocysteine residue. Subcellular fractionation experiments and endoglycosidase H sensitivity indicated that SEPN is a glycoprotein localized within the endoplasmic reticulum. Immunofluorescence analyses confirmed this subcellular localization and GFP fusion experiments demonstrated the presence of an endoplasmic reticulum addressing and retention signal within the N terminus. SEPN was present at a high level in several human fetal tissues and at a lower level in adult ones, including skeletal muscle, which suggests a role for SEPN in early development [23]. 3.3. SEPN1: expression analysis and approach to functional studies A. Lescure developed the approach of his group to analysis of SEPN expression. The trace element selenium is

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an essential nutrient of fundamental importance to human biology. This has become increasingly obvious, as new research has shown an unsuspected role for this trace element in areas important to human health. A link between selenium and muscular pathology was suggested from early observations on animals presenting a deficiency in both selenium and vitamin E. This was then confirmed by the demonstration that mutations in the gene coding for the selenoprotein N cause two forms of early-onset myopathy. The selenoprotein N, SEPN, is a protein of unknown function and no potential biological activity could be derived based on homology to other proteins [24]. In order to better characterize the biological and physiological significance of SEPN, Lescure’s group got interested in its expression profile. First, examination of DNA databases from different animal species revealed that the SEPN is represented in vertebrates only; no homologous protein could be identified in lower organisms or insects. Next, they looked at the tissue-specific expression of SEPN. Surprisingly, studies using nucleotidic probes or antibodies directed against SEPN showed that this protein was homogeneously expressed in all adult tissues examined, making the connection with a muscle-specific disease difficult to interpret. Then, they made the observation that SEPN was more abundantly expressed in fetal tissues compared to adult tissues. This prompted them to study the tissue expression specificity of SEPN during embryogenesis. In situ hybridization on whole zebrafish embryos with a SEPN nucleotidic probe revealed that SEPN transcripts were first detected in the notochord, a precursor of the spine, and then in the somites, from which the skeletal muscles differentiate. Later, during the development, SEPN was more uniformly expressed in all tissues. This observation suggested an involvement of SEPN in early muscle formation, in agreement with the onset of the congenital muscular disease caused by mutations in SEPN1. Presently, different strategies have been undertaken to characterize the molecular and physiological function of SEPN. Identification of a mouse genomic fragment encoding for SEPN allowed this group to design a recombinant DNA molecule to be used to knock-out the SEPN1 gene by homologous recombination in mice. In parallel, they engineered a recombinant SEPN protein that can be produced in large quantity in bacteria. This recombinant protein can be labelled and will be used as a molecular probe to fish-out other protein(s), which physically interact with SEPN. 3.4. Clinical and radiological spectrum of SEPN-related myopathy. Differential diagnosis with MmD/RSMD without SEPN1 mutations Francesco Muntoni reported the clinical and pathological spectrum of 14 cases from 10 families with proven SEPN1 mutations that he has assessed personally. Linkage analysis had been performed in London, while SEPN1 mutation

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analysis had been performed in Paris by Pascale Guicheney. Prof. Caroline Sewry had reviewed the pathology of all cases. Most patients born without contractures had a typical clinical phenotype characterised by early-onset with floppiness, especially poor head control. Most patients acquired independent ambulation with some delay or at a normal age. Common initial concerns were poor head control, stiffness of the cervical spine (invariable in the first decade of life), early-onset scoliosis; in rare instances, the presenting symptom was failure to thrive or respiratory failure. On examination, there was prominent axial weakness predominantly affecting the trunk and neck flexors. In the limbs, the proximal muscles were more affected than the distal muscles. There was mild facial weakness with mid-face hypoplasia, ‘nasal’ tonality of speech and a characteristic ‘bulbous’ nose with nasal bridge prominence. The muscle built was slender without significant muscle hypertrophy, and atrophy of the medial aspect of the thigh muscle was particularly common. Strength remained on the whole stable. A few patients developed mild elbow and Achilles tendon contractures, but significant limb contractures were unusual. Scoliosis was frequently observed, but not invariable: three of the 14 patients did not have scoliosis and in one case there was a severe lordosis affecting the entire spine. Most patients who had developed a scoliosis required surgical stabilisation. A restrictive respiratory syndrome leading to early respiratory failure was common, reflecting a combination of stiffness of the rib cage and diaphragmatic weakness. Most patients needed nocturnal ventilation by their early teens, but some cases as early as 4 years of age. Two patients at the milder end of the spectrum did not require nocturnal ventilation in the late teens/early twenties. All ventilated cases received noninvasive (nasal) ventilation. Intelligence and cardiac function were normal in all cases. Serum CK levels were normal or only mildly elevated. A review of the muscle pathology revealed myopathic changes with no necrosis and little or no regeneration. Endomysial connective tissue was occasionally mildly increased. Uneveness of oxidative enzyme staining or typical minicores were observed in most, but not all biopsies. Brigitte Estournet presented detailed clinical data on the patients with MmD followed at Raymond Poincare´ hospital (Garches). Most of them presented with the classical MmD phenotype, including axial weakness, intercostal muscle contractures, poor thoracic development and early respiratory insufficiency requiring night ventilation. Almost all cases developed a typical scoliosis with dorsal lordosis, particularly progressive at the moment of the growth spurt, requiring arthrodesis. Elbow and sometimes ankle contractures were present from childhood in some of these patients. Homozygous or compound heterozygous SEPN1 mutations have been identified in most of these classical MmD patients, whose phenotype is highly homogeneous. However, other particular features were observed in certain

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families. One of the two affected siblings from a nonconsanguineous family carrying compound heterozygous SEPN1 mutations presented with generalised spinal lordosis and rigidity, while her affected sister showed a typical scoliosis. Another case having a homozygous SEPN1 mutation (1AOG) was initially considered to have an ‘atypical’, particularly severe form of MmD because of early and marked ankle retractions requiring surgical correction, major hyperlordosis from the age of 1 year without scoliosis until the age of 10 years, steady progression of limb weakness and obesity. He lost the ability to walk at 8 years of age, and is only able of partial antigravity movements of hands and forearms at 15 years. He has a facial diplegia and requires permanent ventilation through a tracheotomy, his VC being reduced to 6% in the lying position. On the other hand, no mutation has been found after sequencing of the entire SEPN1 coding sequence in one patient showing a clinical phenotype typical of SEPN-RM; a muscle biopsy could not be performed in this case to verify the morphological diagnosis. B. Estournet presented also the clinical phenotype of two families having the antenatal-onset form of MmD with arthrogryposis. Two siblings from a non-consanguineous Turkish family and one child from unrelated Portuguese parents presented with generalised arthrogryposis including congenital club feet, dorsal kyphosis and dysmorphic features (low-set ears, single palmar crease, oblique palpebral fissures). All showed delayed motor development and muscle weakness predominant in axial and proximal groups. Vital capacity was reduced to 32–67% of the predicted values, but respiratory assistance was not required. Although feet surgery was necessary in all cases, joint contractures improved with intensive physiotherapy. However, the three children developed early kyphosis and/or kyphoscoliosis that was particularly severe and progressive in one of the Turkish brothers; despite a first surgical correction at 4 years, this 11-year-old child needs at present an extensive arthrodesis. Biological material from a second affected foetus in the Portuguese family allowed to exclude linkage of the disease in both families to the SEPN1 locus. Linkage to the RYR1 locus was also excluded in the Portuguese family, but not in the Turkish one, in which RYR1 mutation screen is in progress. Kate Bushby presented clinical and genetic features from seven patients with the ‘classical’ phenotype of MmD under review in Newcastle. The SEPN1 gene had been investigated in five of those cases and one patient was found to be a compound heterozygote for a missense (C461R) and a nonsense (R439X) mutations. This patient had presented with developmental delay and profound axial weakness. Shoulder girdle weakness and wasting, weakness of neck flexion and extension, weakness of small hand muscles and spinal rigidity were prominent findings on examination. There was only mild facial weakness and no external ophthalmoplegia. His further course was characterized by

progressive spinal rigidity and scoliosis, poor weight gain and delayed puberty, and respiratory impairment requiring nocturnal ventilation from the age of 13 years. No SEPN1 mutations could be identified in three clinically almost identical patients, and another patient with overlapping features, but more severe contractures. Two patients have not been investigated yet. Kate Bushby also presented longterm follow-up data of the first case of MmD diagnosed in Newcastle in 1980. This patient presented in infancy with hypotonia and developmental delay, but did not show further progression and at the age of 29 years is still able to walk several miles on the flat. On a recent examination, there was proximal weakness both in hip and shoulder girdles, but no scoliosis or significant cardiorespiratory impairment. The initial biopsy had shown type 1 predominance and multiple cores in all fibres, but a recent repeat biopsy showed no cores, but dystrophic features despite a normal CK, raising the question of the specific diagnosis in this case. Volker Straub presented data on three patients with identified SEPN1 mutations. All three were sporadic cases and showed a typical phenotype, including an early respiratory insufficiency, with vital capacity as low as 28% of the predicted values, which required respiratory support (BiPAP) from the age of 10–14 years. All three cases had a rigid spine syndrome. One of them had also significant elbow contractures and convergent alternating strabismus. Interestingly, a first muscle biopsy from this case, obtained in the neonatal period, had been considered normal. V. Straub pointed out the particular facial appearance of patients with SEPN1 mutations (‘rectangular’ nose, large prominent ears). He also presented the clinical features of cases with a very similar phenotype in which no SEPN1 mutation has been found. Nathalie Goemans presented the clinical features of three ‘classical’ MmD cases with mutations in the SEPN1 gene, one isolated case and two siblings, all of Belgian origin. Muscle biopsies showed the typical pathological features of MmD on light and electron microscopy as described in the previous workshop. These patients were diagnosed at 6, 9 and 6 years and are currently 26, 24 and 21-year-old. All cases presented in the first decade with signs of a mildly progressive myopathy such as difficulties running, hopping and taking stairs. Early motor milestones were achieved within normal range and all walked independently before the age of 18 months. A predominantly axial hypotonia and weakness was an early feature and all patients developed scoliosis, requiring spinal fusion in two cases. Respiratory function dropped during growth spurt to the current vital capacities of 29, 37 and 41% of expected values. Polysomnograhic studies revealed nocturnal hypoventilation and non-invasive nocturnal ventilation was provided in all cases from the age of 12, 16 and 14 years, respectively. After puberty, muscle weakness remained fairly stable. All three patients are currently ambulant for distances between 500 m and several kilometres. Small stature (1), trunk

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deviation, rigidity of the neck, small chest and predominantly axial weakness with nocturnal hypoventilation are the main current symptoms. All three patients lead an independent life and have full-time employment. Genetic analysis of these three cases, published elsewhere [21], revealed a homozygous SEPN1 missense mutation, G315S, in the affected siblings, and a homozygous mutation of the SEPN1 starting codon (1AOG) in the sporadic case. Alan Beggs presented his experience on the genetic and phenotypical analysis of MmD cases collected in the USA. His group studied nine cases belonging to eight families; all showed a variable frequency of minicore-type lesions in their muscle biopsies. Six of these nine cases presented with the classical or moderate form of MmD, the three remaining ones showed atypical clinical findings, including adultonset myalgia without clear muscle weakness. ACTA1 abnormalities were excluded in all these families; RYR1 implication was also excluded in one of the atypical ones. SEPN1 screen, through direct sequencing of the coding region and the entire 3 0 untranslated region, disclosed mutations only in one of the classical MmD cases, who was compound heterozygous for the mutation 997delGTGC/ G943A. This child acquired independent gait at 12 months and was first studied at the age of 3 years because of waddling gait and moderate muscle weakness (4C to 5) in proximal muscles. CK levels were normal. A biceps muscle biopsy at age 3 showed increased variation in muscle fibre size, with normal fibre type distribution; sparse necrotic fibres and mild increase in endomysial and perimysial connective tissue coexisting with multiple oxidative-negative areas on NADH, SDH and COX-stained sections. Utrastructural analysis confirmed the presence of multiple areas devoid of mitochondria, consistent with structured cores. At present, this patient is aged 5 years; no scoliosis or significant reduction of his vital capacity have been observed yet, although he is said to have ‘a mild swayback’. 3.5. Morphological spectrum of SEPN-RM. Immunohistochemical findings Carsten Bo¨nnemann could not attend the meeting, but contributed with a summary of his clinical, morphological and immunohistochemical observations in four patients. The first patient presented with congenital hypotonia and mildly progressive weakness. At 11.5 years, she showed a global amyotrophy, progressive scoliosis with spinal rigidity, proximally pronounced weakness, particularly marked in the neck flexors and respiratory insufficiency. Her phenotype was, thus, consistent with RSMD1, including muscle imaging. Her muscle biopsy showed conspicuous clear zones in the oxidative stains, suggestive of minicore lesions and strongly positive for both filamin C and aB crystalline; as previously reported, these antibodies may reveal core lesions in a non-specific way [25]. The lesions were rare, but large. These findings supported the idea of an

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overlap between RSMD1 and MmD. Three additional patients with very similar histochemical and immunohistochemical findings were identified in short order. In all four patients, ultrastructural studies revealed the presence of minicore lesions in various states in addition to larger zones of myofibrillar disarray. In some of these lesions, filamin C could be demonstrated by immuno-electron microscopy (Prof. H. Goebel, unpublished observations). SEPN1 mutation screening was initiated in Paris for all patients, following the lead of the suggestive phenotype in the first patient. Mutations in SEPN1 were identified in three, but not in the fourth case. The second patient’s phenotype was characterized by breech presentation, congenital hypotonia and weakness, largely non-progressive, and delayed motor development. At 5.5 years, she showed generalised amyotrophy, marked neck flexor weakness, decreased vital capacity, minimal scoliosis, early signs of spinal rigidity and mild hyperlaxity in the upper extremity distal joints. The third patient showed a similar although more severe picture, with congenital hypotonia, delayed motor development, marked muscle weakness predominant in neck flexors, spinal rigidity at 12 years and early respiratory insufficiency requiring nighttime ventilatory support from the age of 6 years. He had an affected brother, reportedly weaker, who died from respiratory insufficiency at the age of 4 years, apparently after suffering from recurrent episodes of respiratory infection. The fourth patient, in which no SEPN1 mutation has been identified, was the product of a gemini pregnancy, born at 30 weeks of gestational age. She presented with delayed motor development and mild, non-progressive muscle weakness, more pronounced proximally and in neck flexors. At 6 years, she had spinal lordosis and mild scoliosis, possibly minimal spinal rigidity, and distal hyperlaxity. Vital capacity was normal. Compared to the other patients in this group, her phenotype was milder, but she showed a more marked joint hyperlaxity. In conclusion, immunohistochemical studies using antibodies directed against filamin C and aB crystalline can be useful to disclose minicore-type lesions in clinically suggestive cases, and therefore support the diagnosis of SEPN-RM and help to direct genetic studies. 3.6. Microarray-based gene expression studies: contribution to nemaline myopathy and preliminary data on SEPN1-related myopathy Alan Beggs presented his experience on microarraybased gene expression studies in congenital myopathies, using the Affymetrix system. This approach was first used by his group at the Children’s Hospital (Boston) for studying nemaline myopathy. Thirteen samples from patients with nemaline myopathy (one due to ACTA1 mutations, none of them carrying TPM3 mutations) were analysed and compared with 21 controls. Transcripts for

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several key enzymes involved in glucose and glycogen metabolism were significantly downregulated, while various structural protein genes, likely associated with fibrosis, were upregulated. Surprisingly, elevated NCAM1 transcription was associated with a 10-fold increase in numbers of satellite cells, despite the fact that nemaline myopathy muscle is not known to be in a regenerative state. A similar approach was used to study a muscle sample from the case with SEPN1 mutations identified by A. Beggs’ group. A muscle sample from this patient was studied using U133, second generation chips, including more than 40,000 probe sets. After a two-cycle amplification of target preparations, modifications of more than five-fold were observed in 86 upregulated probe sets, and in 40, which were downregulated. Upregulated genes included those encoding embryonic isoforms of myosin (MYH3, MYH8) and other developmental proteins such as cardiac ankyrin, as well as some of the extracellular matrix proteins (fibrillin 1, collagens). Similar findings have been observed in other muscle disorders, including dystrophinopathies. An upregulation of heat shock protein 47 gene expression was also observed. In contrast, expression of the gene PSPHL, encoding a phosphoserine-phosphatase like protein, showed significant downregulation. Confirmation and expansion of these exciting preliminary findings will be most helpful to increase our understanding of the pathophysiological consequences of SEPN1 mutations, thereby providing indications about its as far unknown function.

4. Morphological re-evaluation of core myopathies: attempts to differential diagnosis between CCD and MmD Caroline Sewry reviewed and summarized her experience of the muscle pathology of core myopathy cases observed at Oswestry and London. Some of these had identified mutations in the RYR1 gene, some in SEPN1 and others currently remain genetically unresolved. Early descriptions of the pathological features of cases with cores were made before the molecular era, but it is now possible to reassess the pathology in relation to genotype. A few pathological indicators appear to be emerging. Central nuclei can be a feature of central core disease (CCD), but this is not universal and was not common in proven CCD cases from Oswestry, nor in cases with a SEPN1 mutation. Variation in fibre size can occur in CCD, but this is more pronounced in cases with a SEPN1 mutation. Endomysial connective tissue is rare in CCD, although fascicles may be small and separated. In SEPN1 cases, a mild increase in endomysial connective tissue and interstitial fat is common. Type 1 predominance or uniformity is common in CCD, but in SEPN1 cases a two fibre pattern is often preserved. The size of the cores in CCD can be very variable and some cases may not show well-defined cores with standard enzyme techniques (this may relate to age) [26]. Absence of

cores does not exclude a mutation in RYR1. Other cases with RYR1 mutations may show multiple, small cores or unevenness of oxidative enzyme stains. As this is also a feature of cases with SEPN1 mutations, a clear diagnosis based on pathology is not always possible. Large, welldefined cores are not a feature of SEPN1. Electron microscopy shows that areas with sparse mitochondria may be more extensive than areas of myofibrillar disruption, and sometimes only misalignment of myofibrils is observed. Although multiple small areas of myofibrillar disruption occur in cases with SEPN1 mutations, this is not a universal feature. Problems of sampling were discussed and also the application of immunocytochemical methods (in particular studies of gamma filamin and desmin) to identify core-like lesions. Chantal Ceuterick pointed out the fact that muscle samples taken from myotendinous junctions or paravertebral muscles often show non-specific core-like lesions even in healthy subjects; therefore, biopsies performed in these zones are not reliable for the diagnosis of core myopathies.

5. Conclusions/future strategy The second workshop on MmD has demonstrated the rapid advance in the understanding of this condition over the previous 2 years and suggested future lines of enquiry. Phenotype–genotype correlations are gradually emerging, but a precise genetic diagnosis remains difficult and has to be based on a combined assessment of clinical, imaging and histopathological findings. Mutations in RYR1 are responsible for MmD with ophthalmoplegia and for the moderate, ‘CCD-like’ form of MmD. Onset of the muscle symptoms during childhood rather than in the neonatal period, hip dislocation, arthrogryposis, a rather preserved global muscle bulk, pelvic girdle weakness, muscle pain on exertion and generalised joint hyperlaxity are more common in this group of patients, and therefore suggestive of RYR1 involvement. Muscle MRI shows a consistent pattern, particularly in patients harbouring C-terminal mutations, and may further support RYR1 involvement. SEPN1-related myopathy, on the other hand, is characterized by pronounced axial weakness manifesting early as difficulties for head control, while gait is generally acquired at a normal age. Marked amyotrophy, particularly of inner thighs and calves, cervical spine rigidity with scoliosis and severe respiratory impairment contrast with a relatively preserved limb strength and ambulation. Muscle MRI in these patients shows a pattern of selective involvement distinct from the one observed in patients harbouring RYR1 mutations. Anticipation and treatment of respiratory impairment, including regular polysomnographic studies, remains the most important issue in the management of patients with MmD, particularly those forms due to mutations in the SEPN1 gene. Malignant hyperthermia has not been well

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documented in patients with MmD, but has to be anticipated considering the widespread involvement of the RYR1 gene in this condition. Histopathological changes may be highly variable and range from non-specific myopathic changes to the ‘classical’ phenotype of MmD. In RYR1-related cases, type 1 fibre uniformity and a high frequency of centrally located nuclei are often early findings, and cores, although sometimes short on longitudinal sections, on transverse sections tend to be larger than those observed in SEPN1-related myopathies. Biopsy findings may not feature cores at all at an early age, and changes may evolve over time. The morphological spectrum of SEPN-related myopathy is large and may include or not mild dystrophic features, minicore lesions being fairly constant and more easily revealed by ultrastructural and immunohistochemical studies. Whilst SEPN1 mutational analysis is relatively straightforward, screening of the large RYR1 gene remains a daunting task and will have to be limited to the screening of previously identified mutational hotspots in families, where only genomic DNA is available. Screening of cDNA in selected families will hopefully lead to the identification of new mutational hotspots and alleviate the difficulties associated with the large size of the gene in the future. Models for functional studies of both SEPN and RyR1 are emerging and offer the exciting prospect of studying the physiological effects of individual mutations. Finally, RYR1 and SEPN1 involvement has been excluded in approximately 50% of the MmD cases. Recruitment of additional informative families and search for other responsible genes are currently in progress.

Acknowledgements This workshop was made possible thanks to the financial support of the European Neuromuscular Centre (ENMC) and ENMC main sponsors: Association Franc¸aise contre les Myopathies (France), Deutsche Gesellschaft fu¨r Muskelkranke (Germany), Telethon Foundation (Italy), Muscular Dystrophy Group of Great Britain and Northern Ireland (UK), Muskelsvindfonden (Denmark), Prinses Beatrix Fonds (Netherlands), Schweizerische Stiftung fu¨r die Erforschung der Muskelkrankheiten (Switzerland), Verein zur Erforschung von Muskelkrankheiten bei Kindern (Austria), Vereniging Spierziekten Nederland (Netherlands); and ENMC associate member, Muscular Dystrophy Association of Finland.

Appendix A. Workshop participants (Multi-minicore Disease Consortium) Alan Beggs (Boston, USA) Carsten Bo¨nnemann (Go¨ttingen, Germany and Philadelphia, USA)

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Kate Bushby (Newcastle upon Tyne, UK) Chantal Ceuterick-de Groote (Antwerpen, Belgium) Brigitte Estournet-Mathiaud (Garches, France) Ana Ferreiro (Paris, France) Nathalie Goemans (Leuven, Belgium) Pascale Guicheney (Paris, France) Heinz Jungbluth (London, UK) Alain Lescure (Strasbourg, France) Joe¨l Lunardi (Grenoble, France) Francesco Muntoni (London, UK) Ros Quinlivan (Oswestry, UK) Caroline Sewry (London and Oswestry, UK) Volker Straub (Essen, Germany) Susan Treves (Basel, Switzerland)

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