J. Laporte

G.P.39

X-linked myotubular myopathy (XLMTM) is the most severe form of centronuclear myopathy affecting approximately 1 in 50,000 male newborns. Phenotype varies from mild to severe. Several cases of affected females are also reported.presently, there is no effective treatment for the muscle weakness in XLMTM patients. Several promising strategies, including protein replacement therapy and gene therapy, are currently in development.due to the variability of the phenotype, it is important to determine the best outcome measure to assess the efficacy of potential therapies and the prognostic factors for the disease. We present here a prospective study of the pathophysiology of XLMTM to characterize the disease course by using standardized evaluations. A total of 60 patients with XLMTM, male or symptomatic female of any age, are planned to be enrolled in North America and Europe. Visit frequency and assessments are adjusted to age, ambulatory and respiratory status. Evaluations include standard liver ultrasound, clinical exam, ophthalmoplegia assessment, pulmonary function tests, strength and motor function assessment by using upper limb-specific devices and common scales, six-minute walk test, activity monitoring using the Actimyo device and quality of life assessment. Blood samples are collected to test immunization against AAV and to quantify MTM1 protein level. Urinary creatinine excretion is assessed every 6xa0months. Data from the patients’ usual follow-up care are collected from their medical files including psychomotor development, respiratory, cardiac, hematology kidney and liver functions, feeding status and ophthalmologic assessment. According to the weakness of the patient, if needed, the assessments by a physiotherapist can be performed at home. The obtained data will help to characterize the course of disease and the disease spectrum of XLMTM and may help to empower future therapeutic studies as well as to eventually substitute for placebo groups.

G.P.44

We describe extensively the eight patients issued from three French unrelated families with STIM1 mutations, briefly reported in the paper by Bohm et al. (2013). Transmission was autosomal dominant. Onset occurred during childhood (four cases), adolescence (two cases), adulthood (two cases). The first symptom was always related to lower limbs weakness. Examination at age from 21 to 61xa0years showed: (1) weakness predominating in lower proximal muscles; (2) ophthalmoparesis without ptosis in all members from 2 families; (3) significant contractures in 2 families, affecting elbows, wrists, fingers, heel cords, more severe in one patient with neck and masticatory muscles contractures; (4) sparing of facial and bulbar muscles, no specific cardiac involvement. One patient presented a one week long lower limbs paresis at age 9xa0years, a second one reported several days long periods of worsening and a moderate daily variability. The course was mildly progressive in seven patients with preserved ambulation at 50xa0years, no bubar/respiratory involvement. In one patient, evolution was more rapid leading to wheelchair since age of 52xa0years, respiratory insufficiency, swallowing involvement. Muscle imaging revealed fatty degeneration affecting predominantly thighs and posterior legs. Electromyography showed in all cases myogenic patterns and if tested no decrement. Tubular aggregates were found all biopsied patients. A congenital myasthenic syndrome was initially misdiagnosed in several patients due to ophthalmoplegia and variability of symptoms. In conclusion, a characteristic clinical signature was evidenced in our patients associating proximal lower limb weakness, ophthalmoplegia and contractures. Such a phenotype with tubular aggregates at biopsy prompts to look for STIM1 gene mutations.

G.P.264

The congenital myopathies with protein aggregates and inclusions are a subgroup of structural congenital myopathies characterized by aggregation of proteins in muscle. They include Nemaline Myopathy, Core Rod Myopathy, Cap Disease, Reducing body Myopathy, Cylindrical spirals Myopathy, and other entities presenting less distinct protein inclusions. This subgroup represents around 40 % of all biopsies with a structural congenital myopathy. We analysed 154 patients whose muscle was referred to our lab. Pathological diagnosis was achieved combining histochemical and ultrastructural analysis. Nemaline Myopathy was encountered in 103 (67%) patients, 14 (9%) patients had Core Rod myopathy, 12 (8%) Cap Disease, 16 (10%) Reducing Body Myopathy, 3 (2%) Cylindrical spirals Myopathy and 6 (4%) patients presented less distinct protein aggregates. Standard histoenzymology oriented the genetic analysis in around two third of our patients. In one third of cases extended immunohistochemical and ultrastructural studies were pivotal for the constitution of homogenous cohorts, and the orientation of molecular analysis. Genetic diagnosis was achieved in 76 (49%) patients through Sanger sequencing or next generation sequencing. NEB was mutated in 26 patients (34%), ACTA1 in 25 (33%), FHL1 in 14 (18%), TPM2 in 5 (7%) and TPM3 in 3 (4%), RYR1 in 2 (3%), and KLHL40 in 1 (1%). Our study demonstrates that detailed muscle biopsies analysis represents an essential tool in the diagnostic workup of congenital myopathies with protein aggregates and inclusions. Ultrastructural analysis turned to be fundamental in establishing the diagnosis in muscle biopsies from newborn and child patients. A combined clinic-histopathological and genetic approach will certainly help in the identification of the underlying molecular defect in patients without a genetic diagnosis.

G.P.265

Nebulin (NEB) is a sarcomeric structural protein. Mutations in NEB genes particularly cause autosomal recessive nemaline myopathy (NM) and core-rod myopathy. Clinical presentations range from a severe neonatal form to a mild adult-onset form as part of the different diagnosis of distal myopathy. We describe the clinical, histopathological, and molecular findings of three patients with different phenotypes of NEB gene mutations. Case 1: A 5-year-old boy with distal finger retractions, hypotonia, and motor development delay since birth with mainly distal general weakness and hyperlaxity. At age 1 he developed progressive respiratory difficulties needing ongoing invasive ventilation by tracheostomy. He was never able to walk. Spine surgery was performed at 4xa0years. Cognitive development was normal. Quadriceps muscle biopsy showed NM consisting of rod structures, type 1 predominance and atrophy. Exome sequencing showed mutations in exon 139 and 172. Case 2: A 2.5-year-old girl presenting with motor developmental delay, hypomimia, and hypotonia since birth. At 18xa0months of age she walked independently and steppage gait was noticed. She is still improving. Deltoid biopsy showed NM consisting of rod structures and type 1 predominance. Exome sequencing showed mutations in intron 12 and exon 150. Case 3: A 22-year-old girl had frequent falls since she started to walk at 1xa0year, linked to distal hyperlaxity, as well as distal weakness, arreflexia, and steppagge gait. Slow symptom progression was seen. EMG ruled out neuropathic disease and muscle biopsy showed rod and core structures with type 1 predominance. Exome sequencing showed mutations in exon 129 and exon 74 to exon144. Conclusion: NEB-related NM has a broad clinical spectrum particularly including congenital and distal myopathy forms that may mimic a neurogenic process. Our three patients presented with predominantly distal weakness and muscle biopsies showed rod or core-rod myopathies.

G.P.33

In this work we present results of the molecular analyses of 22 families diagnosed with myotubular or centronuclear myopathies (MTM/CNM) on clinical and histological grounds in an outpatient neuromuscular diseases service. Patients were initially screened with focused Sanger sequencing of known genes implicated in MTM/CNM: MTM1 , DNM2 and BIN1 . Out of 8 families with a typical picture of MTM, 7 were diagnosed with known and novel mutations in MTM1 . The remaining 14 families had typical or atypical CNM: two families had DNM2 as the implicated gene, and one family with a single moderately affected female had a macrodeletion in MTM1 demonstrated via MLPA. No mutation was found in BIN1 . A focused panel of 81 genes was performed in two families and allowed a compound TTN truncation, confirmed by Sanger, to be identified as the probable implicated gene in one family. Eleven families without a diagnosis had then whole exome sequencing (WES) performed, followed by a stringent variant filtering pipeline taking into account pathogenicity prediction and population frequency data to filter out polymorphisms. Suitable variants were confirmed with Sanger sequencing. RYR1 was the putative implicated gene in four families, in which mutations were private compound heterozygous, comprising three splice site disruptions and one nonsense. Three additional families with compound missense RYR1 variants with a prediction of high pathogenicity in various tools will need functional validation. The enrichment for RYR1 mutations in this cohort suggests that RYR1 might be a very frequently implicated gene in CNM, especially in cases with atypical histology. Moreover, there does not seem to be a hot spot for mutations in this gene in CNM. Four families still do not have a molecular diagnosis, thus novel genes are probably involved. Ongoing WES sequencing of additional members of these families may help identify novel genes among various candidates.

G.P.38

Centronuclear myopathies (CNM) are congenital muscle disorders characterized by an increased number of muscle fibers with central nuclei. The severe neonatal X-linked form is due to mutations in MTM1, autosomal recessive CNM with neonatal or childhood onset results from mutations in BIN1 (amphiphysin 2), and dominant cases were previously associated to mutations in DNM2 (dynamin 2). However, the genetic basis of the disease is not known for all cases. Here we characterize a novel CNM entity. We established a homogeneous cohort of nine patients from five families with dominant CNM without mutations in DNM2. All patients presented with a mildly progressive adult-onset myopathy and had similar histological and ultrastructural features involving type I fiber predominance and hypotrophy, as well as prominent nuclear centralization and clustering. We identified heterozygous BIN1 mutations in all five families: two missense mutations in the N-terminal amphipathic helix, and three single nucleotide deletions involving the loss of the stop codon. Amphiphysin 2 (encoded by BIN1) induces membrane tubulation and is implicated in excitation-contraction coupling (ECC) at the T-tubules. We confirmed mutation segregation with the disease in the dominant families, and we complemented the molecular diagnosis by functional analyses. Both missense mutations in the N-terminal amphipathic helix strongly decreased the membrane-deforming properties of amphiphysin 2 and three stop-loss mutations resulted in a stable protein containing 52 supernumerary amino acids. Additional immunolabeling experiments revealed abnormal central accumulation of dynamin 2 and caveolin-3, and general membrane alterations of the triad, the sarcolemma, and the basal lamina. In conclusion, we identified BIN1 as a novel gene for dominant adult-onset CNM. Our data provide the evidence that specific BIN1 mutations can cause either recessive or dominant CNM and that both disorders involve different pathomechanisms.

G.P.269

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.

G.P.42

Among congenital myopathies, X-linked recessive myotubular myopathy is one of the most severe forms. The majority of patients present with severe hypotonia at birth and respiratory insufficiency and most of them die in the first year of life. However, there is phenotypic variability. Here, we report two patients carrying a missense mutation in MTM1 gene and presenting with a mild phenotype. The first boy was born on time with respiratory insufficiency and generalised hypotonia. He required ventilator support for 3xa0weeks, and then he went home without specific care. He walked at 11xa0month. He is now 24 and can run, practices canoe and works as a salesperson. At the exam, he presents mild proximal weakness and restrictive syndrome with no ventilator support (VCxa0=xa057%). His brother was born on time with respiratory insufficiency and hypotonia. He stayed in intensive care unit for 6 weeks, then went home without support. He walked at 18xa0months. Respiratory involvement was more severe than his brother with a severe restrictive syndrome (VCxa0=xa033%) but the young man refused non-invasive night-time ventilation proposed at the age of 16. Being now 20, he can walk without any difficulty but cannot run. Clinical exam reveals a mild proximal weakness. Both patients underwent muscle biopsy showing the typical centronuclear myopathy pattern. Molecular analysis of MTM1gene found a hemizygous mutation c.575A>G in exon 8 leading to a missense p.Tyr192Cys. A decreased level of the MTM1 protein was observed by western blot analysis on lymphoblasts. To date, about 300 different mutations in around 540 families have been identified in MTM1 gene, including point mutations, insertions and small and large deletions and duplication. Around 70% of patients presenting with a mild or moderate phenotype carries a missense mutation, which is also the most frequent type of mutation. Another patient has been described with the same mutation (c.575A>G) and presented also with a mild phenotype.

P.9.7 Skeletal muscle biopsy reappraisal in nebulin-related nemaline myopathy

Nemaline myopathy (NM) is a congenital disorder associating muscle weakness with rods in muscle biopsy. Clinical presentation is variable spanning from severe to mild forms. Seven genes are associated with NM. The nebulin (NEB) gene is the most commonly mutated accounting for 50% of cases. We performed a muscle morphological analysis of thirteen NEB-mutated patients with different clinical forms to define pathological patterns and clinical-morphological correlations. Four groups were identified according to clinical severity and age at biopsy. Group 1 ( n xa0=xa04) comprises severe/lethal NM and biopsy in first days. Group 2 ( n xa0=xa03) comprehends severe NM and biopsy after one month. Group 3 ( n xa0=xa03) comprises typical NM and biopsy in childhood, and group 4 ( n xa0=xa02) patients with mild NM and biopsy in adulthood. Molecular diagnosis was done using dHPLC/Sanger-sequencing in six patients and next-generation sequencing in seven. Biopsies underwent histoenzymological, immunohistochemical and ultrastructural analysis. Fiber type distribution, rod characteristics, distribution and localization were investigated. All patients presented NEB mutations consistent with AR inheritance. G1 showed type 2 predominance and scattered squared rods in 1/3 of fibers. Ultrastructural analysis revealed high percentage of fibers with sarcomeric disarray. G2 showed a variable pattern of fiber type distribution spanning from slight type 2 predominance to type 1 uniformity. Rods presented variable distribution and shape. Ultrastructural analysis revealed rare fibers with sarcomeric disarray. In contrast, G3 and G4 presented a homogeneous type 1 uniformity associated with well-delimited subsarcolemmal and/or cytoplasmic elongated rods without sarcomeric alterations. In conclusions we (1) identified an unreported association of type 2 predominance in muscle biopsy of patients presenting severe NEB-related NM; (2) suggest a direct correlation between the association of sarcomeric disarray and clinical severity.

O.22 Autosomal dominant Core Congenital Myopathy caused by a mutation in the MYH7 gene

Autosomal dominant (AD) Central core disease (CCD) named currently congenital myopathy with core is an inherited disorder characterised by the presence of cores which are well-limited rounded areas devoid of any oxidative staining. The cores extend almost along the full length of the fibres; these areas correspond to sarcomeric disorganisation, Z line streaming and absence of mitochondria. Genetic studies of AD-CCD families demonstrated the presence of heterozygous mutations in the RYR1 gene in the large majority of families. However, this gene was excluded in some families, suggesting a genetic heterogeneity of AD-Core myopathies (Romero et al., 2005). To enlarge the genetic spectrum of autosomal dominant congenital myopathies with cores demonstrating mutations in a second gene. AD-CCD family with three affected members: the mother and two of three siblings. The symptoms began during the early childhood with delayed motor development. Later they develop proximal weakness, hypertrophy of calves, scapular winging and significant weakness (amyotrophic) of quadriceps and tibialis anterior. No cardiac or ocular involvement was noted. The muscle biopsies sections showed a particular pattern: relatively large and eccentric cores (placed near the subsarcolemmal regions) associated with type 1 predominance and fibre type disproportion in a patient. The large majority of the cores have abrupt borders. Electron microscopy confirmed the presence of multiple large disorganised sarcomeric areas as characteristic unstructured and structured cores. Moreover, some fibres contain focal disorganised areas. Exome sequencing analysis identified a heterozygous missense mutation L1723P in MYH7 segregating with the disease and affecting a conserved residue in the tail domain. With this study we describe MYH7 as a second causative gene for AD-CCD in addition to RYR1 . Our study enlarges the genetic spectrum of AD-CCD myopathies.