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We report a novel and particularly unusual type of mutation, namely, large deletion in the RYR1 gene, in a Belgian family with myopathy: Patients were found to be compound heterozygous and presented a clinico-pathological phenotype characterized by late-onset and recessive myopathy with cores. We depict the clinical, electrophysiological, pathological and molecular genetic characteristics of family members. To date, large deletions in the RYR1 gene have been reported in only two cases. Both involved different mutations and, in sharp contrast to our cases, presented with a very early-onset, neonatal, and a very severe or lethal phenotype. Overview of reported clinico-pathologic phenotypes, also highlights the rarity of combined late-onset/recessive co-occurrence in this group of myopathies with cores. Finally, this report underlines the broadening spectrum in this group of myopathologic disorders and highlights the concept of 'RYR1-associated/related core myopathies'.

Although the majority (65%) of boys with Duchenne muscular dystrophy (DMD) carry a deletion in the dystrophin gene, finding mutations in the remaining families is vital for counselling. We have provided a comprehensive mutation service as a national referral centre for France for over 10 years and we report here our experience. Mutation screening is on mRNA from a muscle biopsy. We have detected 79 mutations in 89 samples referred with a diagnosis of DMD, which is the most comprehensive survey to date of the full range of nondeletion mutations. Although some mutations were nonsense mutations, some frameshift mutations and some splicing mutations, all of them led to the generation of premature stop codons or a shortened product which could be detected using the Protein Truncation Test. We recommend a protocol which is robust and sensitive applied to the entire coding region reverse-transcribed from dystrophin transcripts from muscle biopsy.

International audience; We report a third patient with typical cap myopathy due to a heterozygous TPM3 mutation, confirming the importance of this causal association. The p.R168C TPM3 mutation we identified has been reported in two previous patients. The histological changes associated with this mutation vary widely from typical cap myopathy with near complete type 1 predominance (two patients), to typical congenital fibre-type disproportion without protein inclusions (one patient). We performed 2D-gel electrophoresis using muscle biopsies from two patients with the p.R168C mutation and show that mutant protein accounts for around 50% of alpha-tropomyosin(slow) in sarcomeres, consistent with a dominant negative mechanism of disease pathogenesis.

CAP-disease is a rare congenital myopathy characterized by the presence of demarcated areas of subsarcolemmal myofibrillar disorganization that are devoid of myosin ATPase, stained with NADH-TR and strongly immunolabeled for desmin. Mutations in ACTA1, TPM2 or TPM3 have been reported so far. We report on 5 cases: 1 sporadic severe case and four patients from 2 families, respectively. In family 1 mother and daughter were affected; in family 2, two siblings manifested the disease. Mutations in the three documented genes were excluded. Age at onset ranged from birth to childhood. The main clinical phenotype consisted of clear dysmorphic features with elongated face and high-arched palate associated with axial, lower and upper limbs muscle weakness. The most severe cases presented early respiratory distress, and feeding difficulties. One patient had arthrogryposis. Muscle biopsies performed at different ages (from newborn to adult age) showed severe changes with marked variation of fiber size, increased internal nuclei, and type I fiber predominance. Several fibers contained a variable number of CAP in subsarcolemal areas. By electron microscopy, well-delineated CAP structures were constantly encountered. These showed disorganization of myofibrils, loss of thick filaments, and thickened fragments of Z bands without nemaline rod bodies. CAP contained segments of sarcomeres constituted by fragments of Z-lines from where arising thin filaments (”butterfly-like” aspect). Furthermore dense thin filamentous material was found in the subsarcolemmal region and less frequently between the myofibrils. In longitudinal sections, the Z lines appeared occasionally jagged and partially misaligned in some myofibrils. No basal membrane abnormalities were observed. To identify the causative mutation whole exome sequencing is ongoing. In conclusion, we attempt to characterize the clinical and morphological particularities of five cases presenting CAP disease, not-related to known genes.

Recurrent rhabdomyolysis episodes in adults are commonly caused by glycogenosis or fatty-acid oxidation disorders. However, in many cases the etiology remains unknown, and recently mutations in RYR1 appeared as a possible cause of exercise induced rhabdomyolysis. We studied 14 adults who presented acute rhabdomyolysis (CK above 10,000 UI/l) triggered by exercise or fever, in whom McArdle disease, fatty acid oxidation disorders, and LPIN1 mutations were excluded. All patients underwent muscle biopsy in order to screen RYR1 mutation on cDNA. Potentially pathogenic mutations in RYR1 gene were detected in 5 unrelated patients. Mutations were the following: p.Val4847Leu, p.G593R, p R3539H, Gly2434Arg, p.Gln3461Pro, p.A1352T, p.A933T and p.Ser1342Gly. Two patients presented each 2 and 3 heterozygous variants. Baseline CK levels were elevated in all cases but one (400–700 UI/l). None of them complained of myalgia or exercise intolerance. Rhabdomyolysis were triggered by weight-training in two patients, viral infection in two cases, and retinoic acid treatment in the last case. Higher recorded CK levels were between 28,000 and 94,000 UI/l, without renal failure. Three patients had a unique episode, whereas two other had several rhabdomyolysis episodes. Muscle biopsies were either normal, or showed moderate structure disorganization. Two siblings of a patient, the father of another patient, and the brother of a third patient, also presented mutations in the RYR1 gene and high baseline CK level. Per-anesthetic malignant hyperthermia was noticed in a grand-mother of one patient. Mutations in RYR1 gene should be systematically searched in patients with exercise or fever induced rhabdomyolysis episodes, after having excluded common metabolic disorders. This diagnostic implies major consequences for genetic counselling in the families, due to the potential risk of anesthetic malignant for the patients and their relatives.

Mutations affecting skeletal muscle isoforms of the tropomyosin genes may cause nemaline myopathy (NM), cap myopathy, core-rod myopathy, congenital fibre-type disproportion, core-rod myopathy, distal arthrogryposes and Escobar syndrome. Here we correlate the clinical picture of these diseases with novel (16) and previously reported (31) mutations of the TPM2 and TPM3 genes. Included are altogether 93 families: 53 with TPM2 mutations and 40 with TPM3 mutations. A total of 27 Twenty-seven distinct pathogenic variants of TPM2, and 20 of TPM3, have been published or listed in the Leiden Open Variant Database ( http://www.dmd.nl/ ). Most are heterozygous changes associated with autosomal dominant disease. Patients with TPM2 mutations tended to present with milder symptoms than those with TPM3 mutations, DA being present only in the TPM2 group. Previous studies have shown that five of the mutations in TPM2 and one in TPM3 cause increased Ca2 + sensitivity resulting in a hypercontractile molecular phenotype. Patients with hypercontractile phenotypes more often had contractures of the limb joints (18/19) and jaw (6/19) than those with non-hypercontractile ones (2/22 and 1/22), while patients with the non-hypercontractile molecular phenotypes more often (19/22) had axial contractures than the hypercontractile group (7/19). Our in silico predictions show that most mutations affect tropomyosin–actin association or tropomyosin head-to-tail binding.

Central Core Disease is a myopathy resulting generally from a mutation in the RYR1 gene, encoding the skeletal muscle calcium release channel RyR1. No treatment is currently available for this disease. We studied a pathological situation in which an affected child harbors two recessive mutations, resulting in a massive reduction in the RyR1 amount. The paternal mutation inducing the inclusion of a new in frame exon in the mRNA of RyR1, resulted in the insertion of additional amino-acids and destabilization of the protein. We hypothesized that inducing the skipping of this exon would be sufficient to restore RyR1 expression and normalization of calcium releases. We developed U7-AON lentiviral vectors to induce exon-skipping on affected primary muscle cells. The efficiency of the exon skipping at the mRNA level, at the protein level and at the functional level using calcium imaging were evaluated. We observed in these affected primary muscle culture a reduction in the inclusion of the additional exon, an increase in the RyR1 protein expression, and a restoration of normal calcium releases. This study is the first demonstration of the potential of exon skipping for the therapy of Central Core Disease, from the molecular to the functional level.

The TPM2 gene encodes beta-tropomyosin (βTm). A range of TPM2 mutations have been associated with several congenital myopathies and distal arthrogryposis. We report a novel dominant TPM2 mutation, K7del, in five unrelated families (de novo occurrence was likely in four families and could be confirmed in two). This mutation removes a highly conserved lysine residue within the N-terminal region that mediates βTm head-to-tail polymerisation. Patients had postnatal large joint contractures and little skeletal muscle weakness until adulthood. Muscle histology showed nemaline bodies and core-like areas, and there was marked fatty infiltration of lower limb muscles by mid-adulthood on muscle MRI. We performed a wide range of studies to investigate the causes of muscle dysfunction. 2D-gel electrophoresis on patient muscle and primary patient cultured myotubes suggested that mutant protein incorporates poorly into sarcomeres and most resides in nemaline rods. Recombinant K7del protein showed a reduced ability to polymerise into long filaments and reduced actin affinity but bound to thin filaments when mixed with wild-type β Tm and caused an in myofibrillar calcium sensitivity. Single dissected patient muscle fibres also showed increased calcium sensitivity of force generation. In summary, K7del is a recurrent TPM2 mutation that causes mild nemaline myopathy with core-like features and postnatal contractures. Mutant K7del protein incorporates poorly into sarcomeres likely due to abnormal head-to-tail polymerisation and reduced actin affinity. Joint contractures may arise from muscle hypercontraction due to increased calcium sensitivity while declining strength in adulthood likely arises from other mechanisms, such as myofibre decompensation and fatty infiltration. These results suggest that patients may benefit from therapies that reduce skeletal muscle calcium sensitivity and we highlight late muscle decompensation as an important cause of morbidity.

G.P.1.01 Phenotypic variations of central core disease O. Paciello , A. Tammaro , L. Passamano , M. Scutifero , E. Picillo , A. Di Martino , S. Papparella , L. Politano University of Naples Federico II, Department of Veterinary Pathology, Naples, Italy, Cardarelli Hospital, Biotechnology Center for Malignant Hyperthermia, Naples, Italy, 3 Second University of Naples, Exp. Medicine – Cardiomyology and Medical Genetics, Naples, Italy, University of Naples Federico II, Department of Veterinary Myology, Naples, Italy