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150th ENMC International Workshop: Core Myopathies, 9–11th March 2007, Naarden, The Netherlands

2008; Elsevier BV; Volume: 18; Issue: 12 Linguagem: Inglês

10.1016/j.nmd.2008.08.001

1873-2364

Heinz Jungbluth, Francesco Muntoni, Ana Ferreiro,

Genetic Neurodegenerative Diseases

1. Introduction and overviewEighteen clinicians and basic scientists from 6 countries convened from the 9th to the 11th of March 2007 in Naarden, The Netherlands for the 150th ENMC sponsored Workshop on Core Myopathies. Members of the ENMC Consortium who attended the 150th ENMC workshop are indicated below: Robert Dirksen, Brigitte Estournet-Mathiaud, Ana Ferreiro, Susan Hamilton, Heinz Jungbluth, Isabelle Marty, Gerhard Meissner, Nicole Monnier, Francesco Muntoni, Ichizo Nishino, Feliciano Protasi, Ros Quinlivan, Caroline Sewry, Volker Straub, Susan Treves, Thomas Voit and Francesco Zorzato.The previous ENMC workshops on Central Core Disease (CCD) in January 2001 and on Multi-minicore Disease (MmD) in May 2000 and November 2002 had lead to collaborations between the participating groups, resulting over the ensuing years in a rapid advance in the understanding of these congenital myopathies.Following identification of mutations in the selenoprotein N (SEPN1) gene as the cause of the most instantly recognizable classic form of MmD [[1]Ferreiro A. Quijano-Roy S. Pichereau C. et al.Mutations of the selenoprotein N gene, which is implicated in rigid spine muscular dystrophy, cause the classical phenotype of multi-minicore disease: reassessing the nosology of early-onset myopathies.Am J Hum Genet. 2002; 71: 739-749Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar], more recently other and clinically widely diverse subgroups of the condition have been associated with recessive mutations in the skeletal muscle ryanodine receptor (RYR1) gene [2Ferreiro A. Monnier N. Romero N.B. et al.A recessive form of central core disease, transiently presenting as multi-minicore disease, is associated with a homozygous mutation in the ryanodine receptor type 1 gene.Ann Neurol. 2002; 51: 750-759Crossref PubMed Scopus (166) Google Scholar, 3Jungbluth H. Müller C.R. Halliger-Keller B. et al.Autosomal-recessive inheritance of RYR1 mutations in a congenital myopathy with cores.Neurology. 2002; 59: 284-287Crossref PubMed Scopus (135) Google Scholar, 4Monnier N. Ferreiro A. Marty I. Labarre-Vila A. Mezin P. Lunardi J. A homozygous splicing mutation causing a depletion of skeletal muscle RYR1 is associated with multi-minicore disease congenital myopathy with ophthalmoplegia.Hum Mol Genet. 2003; 12: 1171-1178Crossref PubMed Scopus (112) Google Scholar, 5Jungbluth H. Zhou H. Hartley L. et al.Minicore myopathy with ophthalmoplegia caused by mutations in the ryanodine receptor type 1 gene.Neurology. 2005; 65: 1930-1935Crossref PubMed Scopus (110) Google Scholar, 6Zhou H. Xu L. Yamaguchi N. et al.Characterization of RYR1 mutations in core myopathies.Human Mol Gen. 2006; 15: 2791-2803Crossref PubMed Scopus (81) Google Scholar], encoding the principal sarcoplasmic reticulum (SR) calcium release channel with a crucial role in excitation–contraction coupling (EC coupling). Because of the marked clinical and histopathologic overlap between RYR1-related recessive MmD and dominantly inherited CCD due to mutations in the same gene, clinical, histopathologic and genetic features of both conditions were covered in the present workshop on core myopathies. With the genetic background of MmD and CCD now largely established, a strong emphasis of this workshop was placed on the physiology and pathophysiology of intracellular calcium metabolism and of EC coupling associated with mutations in the RYR1 and SEPN1 genes, and animal models of those conditions which may provide a basis for future therapeutic interventions.After the welcoming address by Pascale Guicheney on behalf of Kate Bushby, Research Director of the ENMC, the workshop was introduced by Ana Ferreiro and Heinz Jungbluth, who gave an overview over developments since the most recent workshop on MmD in November 2002.Mutations in the skeletal muscle ryanodine receptor (RYR1) gene, initially associated with the dominantly inherited malignant hyperthermia susceptibility (MHS) trait and the congenital myopathy central core disease (CCD), have now been associated with a wide range of congenital myopathy phenotypes including subgroups of Multi-minicore Disease (MmD) [1Ferreiro A. Quijano-Roy S. Pichereau C. et al.Mutations of the selenoprotein N gene, which is implicated in rigid spine muscular dystrophy, cause the classical phenotype of multi-minicore disease: reassessing the nosology of early-onset myopathies.Am J Hum Genet. 2002; 71: 739-749Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 3Jungbluth H. Müller C.R. Halliger-Keller B. et al.Autosomal-recessive inheritance of RYR1 mutations in a congenital myopathy with cores.Neurology. 2002; 59: 284-287Crossref PubMed Scopus (135) Google Scholar, 4Monnier N. Ferreiro A. Marty I. Labarre-Vila A. Mezin P. Lunardi J. A homozygous splicing mutation causing a depletion of skeletal muscle RYR1 is associated with multi-minicore disease congenital myopathy with ophthalmoplegia.Hum Mol Genet. 2003; 12: 1171-1178Crossref PubMed Scopus (112) Google Scholar, 5Jungbluth H. Zhou H. Hartley L. et al.Minicore myopathy with ophthalmoplegia caused by mutations in the ryanodine receptor type 1 gene.Neurology. 2005; 65: 1930-1935Crossref PubMed Scopus (110) Google Scholar, 6Zhou H. Xu L. Yamaguchi N. et al.Characterization of RYR1 mutations in core myopathies.Human Mol Gen. 2006; 15: 2791-2803Crossref PubMed Scopus (81) Google Scholar] and centronuclear myopathy (CNM) [[7]Jungbluth H. Zhou H. Sewry C.A. et al.Centronuclear myopathy due to a de novo dominant mutation in the skeletal muscle ryanodine receptor (RYR1) gene.Neuromuscul Disord. 2007; 17: 338-345Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar]. Whilst typical dominantly inherited CCD has been associated with a fairly mild phenotype characterized by hip girdle weakness and absence of overt bulbar, extraocular and respiratory involvement, more severe presentations and diverse clinical features including arthrogryposis, external ophthalmoplegia, substantial bulbar involvement and respiratory compromise have been recently reported and predominantly attributed to recessively inherited RYR1 mutations. Corresponding to the more generalized clinical muscle involvement, recessive RYR1 mutations are associated with a more diffuse increase in signal intensity on muscle MRI of the lower limb [[5]Jungbluth H. Zhou H. Hartley L. et al.Minicore myopathy with ophthalmoplegia caused by mutations in the ryanodine receptor type 1 gene.Neurology. 2005; 65: 1930-1935Crossref PubMed Scopus (110) Google Scholar] compared to the highly selective pattern observed in patients with dominant RYR1 mutations [[8]Jungbluth H. Davis M.R. Muller C. et al.Magnetic resonance imaging of muscle in congenital myopathies associated with RYR1 mutations.Neuromuscul Disord. 2004; 14: 785-790Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar]. Whilst dominant RYR1 mutations associated with a typical CCD phenotype localize almost exclusively to the RYR1 C-terminus, predominantly exons 100–101, recessive RYR1 mutations appear widespread throughout the RYR1 coding sequence but more extensive data are currently only emerging. More recently, expression of heterozygous mutations on a haploinsufficient background due to epigenetic allele silencing of the RYR1 gene has been reported as a novel mechanism in the pathogenesis of core myopathies [[9]Zhou H. Jungbluth H. Sewry C.A. et al.Molecular mechanisms and phenotypic variation in RYR1-related congenital myopathies.Brain. 2007; 130: 2024-2036Crossref PubMed Scopus (127) Google Scholar]. Whilst the pathogenesis of MH and CCD has been extensively investigated, functional studies on MmD-related RYR1 mutants are currently still limited but suggest a wider range of underlying mechanisms.2. Histopathologic spectrum and differential diagnosis of core myopathiesCaroline Sewry gave an overview of the pathological changes in muscle biopsies from 28 cases with mutations in the RYR1 gene and 9 cases with mutations in the SEPN1 gene, based on the experience at the Hammersmith Hospital Neuromuscular Unit and Robert Jones & Agnes Hunt Hospital, Oswestry. Caroline Sewry emphasised that cores per se are not specific and can be seen to varying degrees in several neuromuscular disorders, including muscular dystrophies and neurogenic disorders, where they sometimes appear as targets with a pronounced rim. Seventeen of the RYR1 cases had autosomal dominant inheritance (11 de novo), 5 cases had recessive inheritance, and an additional 6 cases had haploinsufficiency with monoallelic RyR1 expression in muscle. The SEPN1 cases had homozygous or heterozygous recessive mutations.Large classical cores lacking oxidative enzyme stains and extending an appreciable length down the fibre were present in several of the dominant RYR1-related cases, particularly those with mutations in the ‘hot spot’ RYR1 C-terminal region; the position of these cores may be central, peripheral or eccentric, even within the same section. The spectrum associated with the same mutation may be wide, as demonstrated in three individuals from the same family: at 4 months of age the female proband showed no cores, whilst her 3-year-old brother showed large classical cores and the mother unevenness of stain. The absence of cores may relate to age and this needs to be considered when assessing biopsies. Recessive RYR1 mutations seemed to be more frequently associated with focal small multiple cores (minicores) or large focal cores that may extend across the width of the fibre, the latter appearance often seen in cases with ophthalmoplegia; the distinction between a ‘minicore’ or ‘central core’ is therefore not always clear and the term ‘core myopathy’ is now often used by the Hammersmith and Oswestry groups.Ultrastructurally, cores in most RYR1 cases are of the structured type, with moderate preservation of myofibrillar structure; in occasional cases, however, they may be mainly of the unstructured type lacking ATPase activity. Cores lack phosphorylase, glycogen and mitochondria, and the area without the latter may be more extensive than the area of disrupted myofibrils. Some cores may be rimmed by glycogen, mitochondria and various proteins and/or show accumulation of proteins, including desmin, γ-filamin, syncoilin, myotilin, SERCA2, calsequestrin, dihydropyridine receptor and RyR1 protein. Phalloidin binding often highlights the hypercontraction of the myofibrils rather than an accumulation of F-actin. The accumulation of proteins and rimming of the cores varies between cores, even within one biopsy and no consistent patterns were observed in this series of cases.In addition to core structures, most RYR1 cases show pronounced slow/type 1 fibre type predominance or uniformity, with the majority of fibres expressing slow myosin and only a few fibres co-expressing slow and fast myosin isoforms. Variation in fibre size is often not marked in RYR1 cases but very small fibres (often <5 μm) expressing neonatal myosin are sometimes seen. The presence of adipose tissue is common and may be extensive; in some cases it may also be associated with an increase in fibrous tissue and resemble a congenital muscular dystrophy, but there is usually no fibre necrosis. Sampling of muscles with varying degrees of differential involvement may result in variable amounts of adipose and fibrous tissue in a biopsy.Internal nuclei, some of which are central or multiple, are also a common feature; the presence of central nuclei can resemble myotubular/centronuclear myopathy and the RYR1 gene should be considered when this pathology is present, in addition to the MTM1, DNM2 and DM1 genes typically associated with this appearance. Internal nuclei are also common at myotendinous junctions and fibres with multiple splits resembling these areas are common in RYR1 cases, although they are not specific. Some cases may show clusters of nemaline rods in occasional fibres but these are not marked features. No cases in this series with proven mutations resembled the core-rod myopathy cases reported in the literature.In comparison, cases with SEPN1 mutations often show less marked pathology with preservation of the two-fibre pattern, although moderate type 1 predominance can occur. Unevenness of oxidative enzyme stains or focal cores are present in both fibre types. Internal nuclei may also occur but are not usually as pronounced as in RYR1 cases. Similarly, a mild or moderate increase in adipose and endomysial connective tissue may occur but these are not as extensive as in RYR1 cases. The focal cores in SEPN1-related cases are not rimmed by accumulation of proteins or glycogen, but accumulation of some proteins may occur in some fibres, for example desmin and/or myotilin.Ana Ferreiro presented morphological and immunohistochemical data from the core myopathy biopsies analysed at the Institut de Myologie (Paris). In RYR1-related core myopathies, most of the findings were fully consistent with those observed in the Hammersmith and Oswestry series described above. Well-delimited or rimmed cores, more often peripheral and multiple than central and unique, running most of the length of type 1 fibres, were highly characteristic of dominant CCD due to heterozygous RYR1 mutations, and have never been observed in classical, recessive MmD due to SEPN1 mutations. Conversely, short, poorly defined minicores were associated with dominant or recessive RYR1 mutations as well as with recessive SEPN1 mutations. Furthermore, age-related morphological evolution from minicores to cores has been documented on sequential deltoid biopsies from the same patient with a homozygous RYR1 mutation [[2]Ferreiro A. Monnier N. Romero N.B. et al.A recessive form of central core disease, transiently presenting as multi-minicore disease, is associated with a homozygous mutation in the ryanodine receptor type 1 gene.Ann Neurol. 2002; 51: 750-759Crossref PubMed Scopus (166) Google Scholar]. These findings underline that, in RYR1-related myopathies, there is a morphological continuum between minicores and cores, and support the global denomination of “core myopathies” for this group of congenital muscle disorders. The form of MmD with ophthalmoplegia associated with homozygous RYR1 mutations often showed cores which were short on the longitudinal axis but had a large transversal diameter (spanning most of the transverse fibre section) with abundant central nuclei [[4]Monnier N. Ferreiro A. Marty I. Labarre-Vila A. Mezin P. Lunardi J. A homozygous splicing mutation causing a depletion of skeletal muscle RYR1 is associated with multi-minicore disease congenital myopathy with ophthalmoplegia.Hum Mol Genet. 2003; 12: 1171-1178Crossref PubMed Scopus (112) Google Scholar].A retrospective analysis of 42 muscle biopsies from patients with homozygous or compound heterozygous mutations of the SEPN1 gene confirmed that several categories of morphological lesions can be associated with SEPN1-related myopathy. Both fibre types were usually present; type 1 predominance and relative hypotrophy (fibre type disproportion) were observed in 97% cases. Most samples (83.3%) showed multiple minicores in type 1 and type 2 fibres, either isolated (45%), associated with dystrophic findings (21%), or with Mallory body-like inclusions (MBs, 17%). When present, dystrophic lesions were generally mild. MBs (hyaline inclusions containing desmin, with three components on EM) affected usually less than 10% of the fibres. There was no correlation between the morphological pattern and the type or localization of SEPN1 mutations. To some extent, morphological patterns correlated with the muscle biopsied: minicores and dystrophic lesions were present in all the muscles sampled, but a dystrophic pattern was more common in axial muscles.Several proteins such as desmin, α B crystalline or γ filamin accumulate in cores, minicores and targets, in a non-specific way. To search for distinctive markers, the immunolocalization of six proteins of the Ca2+-release complex was analysed in the biopsies from 12 genetically-characterized core myopathy cases. In 7 cases with RYR1 mutations (6 CCD, 1 MmD), RyR1 was depleted from the cores; in contrast, the other proteins of the sarcoplasmic reticulum (calsequestrin, SERCA1/2 and triadin) and the T-tubule (dihydropyridine receptor-α1 subunit) were accumulated within or around the lesions, suggesting an original modification of the Ca2+-release complex protein arrangement. Conversely, all Ca2+-related proteins were distributed normally in 5 MmD cases with SEPN1 mutations. These results provide an appropriate tool to orientate the differential and molecular diagnosis of core myopathies, and suggest that different pathophysiological mechanisms lead to core formation in SEPN1- and in RYR1-related core myopathies.Ichizo Nishino presented the pathological spectrum of three RYR1-related conditions in a large cohort of Japanese patients: malignant hyperthermia susceptibility (MHS), central core disease (CCD) [[10]Wu S. Ibarra M.C. Malicdan M.C. et al.Central core disease is due to RYR1 mutations in more than 90% of patients.Brain. 2006; 129: 1470-1480Crossref PubMed Scopus (159) Google Scholar] and congenital neuromuscular disease with uniform type 1 fibres (CNMDU1). In the Japanese cohort both CCD with typical pathological features and CNMDU1 were associated with C-terminal mutations; in contrast, all cases with atypical cores and the majority of patients with MHS had mutations in non-C-terminal regions. In addition, isolated single fibre analysis in two patients with typical CCD with C-terminal mutations did not reveal increased calcium-induced calcium release (CICR), suggesting that, albeit limited number of cases, some CCD causing C-terminal mutations may not be associated with MHS. Interestingly, histopathologic changes in one of the few C-terminal mutations associated with MHS, p.A4894T, were characterized by a normal mosaic pattern of fibre types and core-like structures in only a few fibres; in contrast, p.A4894P was associated with CNMDU1, suggesting that substitution to a different amino acid, even at the same position, may result in a different phenotype.3. Clinical phenotype of core myopathies with or without RYR1 mutationsRos Quinlivan presented clinical features of nine patients from five families with Multi-minicore Disease but no confirmed SEPN1 or RYR1 mutation. Clinical features in those families included: arthrogryposis with distal involvement in the upper limbs, adducted thumbs or camptodactyly, and ptosis; cleft palate was present in two unrelated children. In two families rigid spine was a feature associated with restrictive respiratory insufficiency, one child has had scoliosis surgery. Muscle weakness was mild predominantly affecting truncal and pelvic girdle muscles. Two children had mild educational difficulties and one had epilepsy. One consanguineous family presented with a different phenotype affecting the mother and two children. Clinical features included: ophthalmoplegia, ptosis and scoliosis associated with generalized muscle atrophy and poor feeding. Electromyography revealed myopathic features. All of the patients have normal CK, there was no cardiac involvement and none of the patients to date required non-invasive ventilation. Those findings suggest either non-exonic mutations in the SEPN1/RYR1 genes or genetically distinct phenocopies of patients with mutations in above genes.Heinz Jungbluth and Francesco Muntoni presented 3 children from two unrelated families with clinical and histopathologic features of a RYR1-related congenital myopathy but without confirmed RYR1 mutation. Heinz Jungbluth reported a 7-year-old boy who presented from birth with hypotonia, bulbar involvement and arthrogryposis following a pregnancy complicated by oligohydramnios and reduced fetal movements; his further course was characterized by developmental delay and slow progression. On examination, distribution of weakness and wasting pronounced in the axial and shoulder girdle muscles was similar to that observed in patients with recessive RYR1 mutations. Muscle biopsy showed marked type 1 predominance, increase in internal nuclei and unevenness of oxidative stain. Although no mutation could be detected on sequencing of the entire RYR1 coding sequence, RyR1 protein was markedly reduced on Western blotting.Volker Straub presented patients from the neuromuscular group in Newcastle that showed clinical symptoms and muscle biopsy findings suggestive of CCD or MmD but without mutations in the SEPN1 gene or mutations in exons 95, 100, 101 and 102 of the RYR1 gene. In addition to weakness of the axial muscles several patients showed clinical symptoms that are not classically associated with CCD or MmD. A now 12-year-old boy with characteristic findings of CCD in his muscle biopsy, congenital onset of weakness and respiratory insufficiency showed marked distal hyperlaxity that resembled patients with Ullrich congenital muscular dystrophy (UCMD); however, no abnormalities of collagen VI expression in the patient’s muscle biopsy or in his fibroblasts were detected. It was felt that there is a clear clinical overlap between patients with CCD, MmD and UCMD. A 24-year-old lady with cores in her biopsy, generalized weakness and rigidity of her spine presented with slurred speech that has previously been described in patients with myofibrillar myopathies; although the biopsy did not show vacuoles or desmin accumulation there may well be an overlap between patients with core diseases and myofibrillar myopathies. A 35-year-old patient with congenital onset of weakness and cores in his biopsy (confirmed on electron microscopy) presented with external ophthalmoplegia and excessive sweating (hyperhydrosis); he also experienced difficulties with temperature regulation. It is not clear whether mutations in the RYR1 gene can affect the autonomous nervous system. Additional features in other patients with a CCD/MmD phenotype but no mutation in either the SEPN1 gene or the common exons of the RYR1 gene were learning difficulties; all of these patients had a normal serum CK activity4. Genetic diagnosis of RYR1-related core myopathiesNicole Monnier presented genetic findings in a French cohort of 229 unrelated patients presenting with core myopathies ranging from congenital onset with severe phenotype to milder classical CCD. Muscle biopsies from those patients were characterized by core lesions showing variable localization, size, length and number within the muscle fibres and by frequent association with centrally located nuclei and predominance of type I fibres. RYR1-related diseases including malignant hyperthermia (MH) with cores, exertional heat stress syndrome and centronuclear myopathies were excluded. Most cases were sporadic (156) while the remaining patients had a family history of dominant (59) or recessive (13) core myopathy. Search for mutations in the RYR1 gene was performed by C-terminal screening of exons 92 to 106 and, when a muscle biopsy was available, by cDNA sequencing in 52 cases.A single dominant mutation was identified in 54 cases, two recessive mutations each in 27 cases and one mutation of unknown significance in 20 cases. Furthermore, no mutation was identified after cDNA sequencing in 9 cases.RYR1 mutations identified in dominant core myopathies (n = 61) were missense mutations and in-frame deletions localized in the C-terminal domain of the protein, mostly affecting transmembrane and luminal domains of the calcium channel. Sixty percent of those mutations were recurrent and 26% were neomutations.In contrast, RYR1 mutations identified in recessive core myopathies (n = 54) were distributed along the entire coding sequence of the gene. Seventy seven percent were missense and in-frame deletions while 23% were truncating mutations leading to loss of protein (Tr). Three cases were compound heterozygous for 2 Tr mutations associated with strong RyR1 depletion. All patients presented with a severe neonatal phenotype. Eight cases were compound heterozygous for 1 Tr mutation and 1 missense mutation resulting in hemizygous expression of the mutated RyR1 protein. The variability observed in phenotype severity ranging from mild to severe was likely to be related to the nature of the missense mutation. The remaining 14 patients were homozygous (n = 2) or compound heterozygous (n = 12) for missense mutations that included a striking high frequency of MH mutations. We have also identified a recessive case of severe central core myopathy in a dominant CCD family.Francesco Muntoni presented genetic findings in a large cohort of core myopathy patients from the United Kingdom [[9]Zhou H. Jungbluth H. Sewry C.A. et al.Molecular mechanisms and phenotypic variation in RYR1-related congenital myopathies.Brain. 2007; 130: 2024-2036Crossref PubMed Scopus (127) Google Scholar]. In order to characterize the spectrum of congenital myopathies associated with RYR1 mutations, his group investigated a cohort of 44 patients from 28 families with clinical and/or histopathologic features suggestive of RYR1 involvement, and identified 25 RYR1 mutations, 9 of them novel, including 12 dominant and 13 recessive mutations. With only one exception, dominant mutations were associated with a CCD phenotype, prominent cores, and predominantly occurred in RYR1 C-terminal exons 101 and 102, whereas the 13 recessive RYR1 mutations were distributed evenly along the entire RYR1 gene and were associated with a wide range of clinico-pathological phenotypes. Protein expression studies in 9 cases suggested a correlation between specific mutations, RyR1 protein levels and resulting phenotype: in particular, whilst patients with dominant or some recessive mutations and typical CCD phenotypes appeared to have normal RyR1 expression, individuals with more generalized weakness, multi-minicores and external ophthalmoplegia had a severe depletion of the RyR1 protein. The phenomenon of severe protein depletion was observed in patients compound heterozygous for a recessive mutation and an apparently normal but silenced allele, providing evidence for the pathogenic role of allele silencing at the RYR1 locus when associated with recessive RYR1 mutations. These data indicate complex genotype–phenotype correlations associated with RYR1 mutations differentially affecting assembly and function of the RyR1 calcium release channel.Heinz Jungbluth presented findings in a cohort of patients with a core myopathy associated with monoallelic expression of a heterozygous RYR1 missense mutation due to epigenetic allele silencing of the second RYR1 allele [[6]Zhou H. Xu L. Yamaguchi N. et al.Characterization of RYR1 mutations in core myopathies.Human Mol Gen. 2006; 15: 2791-2803Crossref PubMed Scopus (81) Google Scholar]. These patients had consistent clinical features characterized by external ophthalmoplegia and predominant axial and proximal weakness; muscle biopsies were characterized by marked type 1 uniformity and core-like structures often spanning the entire fibre diameter on longitudinal sections. Haplotyping studies in those families were consistent with recessive inheritance, however, sequencing of the entire RYR1 coding sequence in patients demonstrated a single RYR1 missense mutation invariably inherited from an unaffected father. Whilst these mutations were expressed heterozygously on the genomic DNA level, studies on cDNA derived from skeletal muscle tissue showed monoallelic expression of the mutated RYR1 allele, consistently due to maternal allele silencing and suggestive of genomic imprinting. Genomic imprinting is an epigenetic phenomenon resulting in allele-specific silencing according to parental origin; this may be tissue-specific, developmentally regulated and also polymorphic in the general population. Tissue-specific allele silencing was implicated by failing to identify monoallelic RYR1 expression in skin fibroblasts or lymphocytes from the same patients; further studies on normal fetal tissues suggested tissue-specific RYR1 allele silencing in skeletal muscle, brain, spinal cord, eye and intestine in 10% of a normal population which was not present in adult skeletal muscle. Reactivation of the silenced maternal allele by the DNA methyltransferase inhibitor 5′-aza-deoxycytidine (5-azaC) in cultured skeletal muscle myoblasts from patients implicated DNA hypermethylation as the underlying mechanism, however, bisulfite sequencing failed to show the differentially methylated region (DMR) within the 5′ region of the RYR1 gene, suggesting a control region further afield. These results suggested that RYR1 is polymorphically silenced in a tissue-specific manner during human fetal development, and that in a proportion of recessive core myopathy patients, the silencing of one RYR1 allele contributes to unmask the phenotype in the presence of recessive paternal mutations. Whilst consistent silencing of the maternal allele in the original cohort of patients strongly implicated imprinting as the mechanism underlying RYR1 allele silencing, more recent identification of a patient with a maternally inherited RYR1 mutation and silencing of the paternal allele indicates the existence of alternative mechanisms with similar effect.5. Calcium handling and excitation–contraction coupling: Proteins involved and animal modelsFeliciano Protasi presented his work on spatial relationships between the key proteins of excitation–contraction EC coupling, the skeletal muscle ryanodine receptor (RyR1) and the dihydropyridine receptor (DHPR), in skeletal muscle. In muscle fibres, an extremely well organized system of tubules and vesicles, collectively named sarcotubular system, is able to finely control the cytoplasmic Ca2+ concentration. The sarcotubular system is formed by two separate systems of membranes: exterior membranes (sarcolemma and its invaginations,