T. Cullup

RYR1 mutations are a common cause of congenital myopathies with central nuclei.

Centronuclear myopathy (CNM) is a rare congenital myopathy characterized by prominence of central nuclei on muscle biopsy. CNM has been associated with mutations in MTM1, DNM2, and BIN1 but many cases remain genetically unresolved. RYR1 encodes the principal sarcoplasmic reticulum calcium release channel and has been implicated in various congenital myopathies. We investigated whether RYR1 mutations cause CNM.

Impaired neuromuscular transmission and response to acetylcholinesterase inhibitors in centronuclear myopathies

Many clinical features of autosomal centronuclear myopathies (CNM) and X-linked myotubular myopathy (XLMTM) are common to congenital myasthenic syndromes (CMS). We describe three children whose clinical and electrophysiological findings originally suggested CMS, in whom CNM was diagnosed pathologically, though not yet genetically characterised. A fourth case, with XLMTM, also showed electrophysiological features of a neuromuscular transmission defect. Three (including the XLMTM case) showed improved strength with acetylcholinesterase inhibitor treatment. We also studied neuromuscular junction structure and function in the MTM1 knockdown zebrafish model of XLMTM, demonstrating abnormal neuromuscular junction organization; anticholinesterase therapy resulted in marked clinical response. These observations suggest that a neuromuscular transmission defect may accompany CNM and contribute to muscle weakness. Muscle biopsy should be considered in infants suspected to have CMS, especially if treatment response is incomplete, or no CMS gene mutation is identified. Treatment with acetylcholinesterase inhibitors may benefit some CNM patients. This warrants further confirmation.

Congenital myopathies - clinical features and frequency of individual subtypes diagnosed over a 5-year period in the United Kingdom

The congenital myopathies are a group of inherited neuromuscular disorders mainly defined on the basis of characteristic histopathological features. We analysed 66 patients assessed at a single centre over a 5 year period. Of the 54 patients where muscle biopsy was available, 29 (54%) had a core myopathy (central core disease, multi-minicore disease), 9 (17%) had nemaline myopathy, 7 (13%) had myotubular/centronuclear myopathy, 2 (4%) had congenital fibre type disproportion, 6 (11%) had isolated type 1 predominance and 1 (2%) had a mixed core-rod myopathy. Of the 44 patients with a genetic diagnosis, RYR1 was mutated in 26 (59%), ACTA1 in 7 (16%), SEPN1 in 7 (16%), MTM1 in 2 (5%), NEB in 1 (2%) and TPM3 in 1 (2%). Clinically, 77% of patients older than 18 months could walk independently. 35% of all patients required ventilatory support and/or enteral feeding. Clinical course was stable or improved in 57/66 (86%) patients, whilst 4 (6%) got worse and 5 (8%) died. These findings indicate that core myopathies are the most common form of congenital myopathies and that more than half can be attributed to RYR1 mutations. The underlying genetic defect remains to be identified in 1/3 of congenital myopathies cases.

Mutations in MYH7 cause Multi-minicore Disease (MmD) with variable cardiac involvement

Central Core Disease (CCD) and Multi-minicore Disease (MmD) (the core myopathies) have been mainly associated with mutations in the skeletal muscle ryanodine receptor (RYR1) and the selenoprotein N (SEPN1) gene. A proportion of cases remain unresolved. Mutations in MYH7 encoding the beta myosin heavy chain protein have been implicated in cardiac and, less frequently, skeletal muscle disorders. Here we report four patients from two families with a histopathological diagnosis of MmD, presenting in childhood with slowly progressive muscle weakness, more proximal in Family 1 and more distal in Family 2, and variable degrees of cardiorespiratory impairment evolving later in life. There was also a strong family history of sudden death in the first family. Muscle biopsies obtained in early childhood showed multiple minicores as the most prominent feature. Sequencing of the MYH7 gene revealed heterozygous missense mutations, c.4399C>G; p.Leu1467Val (exon 32) in Family 1 and c.4763G>C; p.Arg1588Pro (exon 34) in Family 2. These findings suggest MYH7 mutations as another cause of a myopathy with multiple cores, in particular if associated with dominant inheritance and cardiac involvement. However, clinical features previously associated with this genetic background, namely a more distal distribution of weakness and an associated cardiomyopathy, may only evolve over time.

Relative frequency of congenital muscular dystrophy subtypes: analysis of the UK diagnostic service 2001-2008.

The Dubowitz Neuromuscular Centre is the UK National Commissioning Group referral centre for congenital muscular dystrophy (CMD). This retrospective review reports the diagnostic outcome of 214 UK patients referred to the centre for assessment of possible CMD between 2001 and 2008 with a view to commenting on the variety of disorders seen and the relative frequency of CMD subtypes in this patient population. A genetic diagnosis was reached in 53 of 116 patients fulfilling a strict criteria for the diagnosis of CMD. Within this group the most common diagnoses were collagen VI related disorders (19%), dystroglycanopathy (12%) and merosin deficient congenital muscular dystrophy (10%). Among the patients referred as possible CMD that did not meet our inclusion criteria, congenital myopathies and congenital myasthenic syndromes were the most common diagnoses. In this large study on CMD the diagnostic outcomes compared favourably with other CMD population studies, indicating the importance of an integrated clinical and pathological assessment of this group of patients.

Congenital fibre type disproportion associated with mutations in the tropomyosin 3 (TPM3) gene mimicking congenital myasthenia

Congenital myopathy with fibre type disproportion (CFTD) has been associated with mutations in ACTA1, SEPN1, RYR1 and TPM3 genes. We report the clinico-pathological and electrophysiological features of 2 unrelated cases with heterozygous TPM3 mutation. Case 1 is a 19-year-old lady who presented with motor delay in infancy, respiratory failure in early teens requiring non-invasive ventilation despite being ambulant, ptosis, axial more than proximal weakness and scoliosis. Case 2 is a 7-year-old boy with hypotonia, feeding difficulties, motor delay and scoliosis, also requiring non-invasive ventilation while ambulant. Muscle biopsies in both cases showed fibre type disproportion. Muscle MRI (Case 1) showed mild uniformly increased interstitial tissue in and around the muscles. Sequencing of TPM3 in case 1 revealed a previously described heterozygous c.503G > A(pArg168His) missense variant in exon 5 and a novel heterozygous missense mutation c.521A > C(pGlu174Ala), also in exon 5, in case 2. A mild abnormality in the single fibre EMG was documented on electrophysiology in both cases. These cases highlight the neuromuscular transmission defect in CFTD secondary to TPM3 mutations.

Centronuclear myopathy with cataracts due to a novel dynamin 2 (DNM2) mutation

Dynamin 2 (DNM2)-related dominant centronuclear myopathy is usually a mild disorder, but more severe variants have been associated with mutations affecting the pleckstrin homology (PH) domain of the protein, mainly implicated in different forms of Charcot-Marie-Tooth Disease (CMT). Whilst DNM2-related CMT may feature non-neurological findings including cataracts, this has not been reported in DNM2-related centronuclear myopathy. We report a girl presenting from birth with hypotonia, respiratory and feeding difficulties. Motor development was delayed and at 9years she lost the ability to walk. She had ptosis, external ophthalmoplegia and bilateral cataracts. Muscle biopsy showed increase in central nuclei with type 1 hypotrophy and fibrosis. DNM2 screening revealed a novel heterozygous substitution (c.1862T>C; p.Leu621Pro) affecting the PH domain of the protein. Her further course was progressive and at 14years she died from respiratory failure. Our findings expand the phenotypical spectrum associated with DNM2 mutations and provide a new clinical indicator for involvement of this gene in patients with centronuclear myopathy.

X-linked myotubular myopathy due to a complex rearrangement involving a duplication of MTM1 exon 10

X-linked myotubular myopathy is a predominantly severe congenital myopathy with central nuclei on muscle biopsy due to mutations in the MTM1 gene encoding myotubularin. We report a boy with typical features of X-linked myotubular myopathy. Sequencing of the MTM1 gene did not reveal any causative mutations. Subsequent MLPA analysis identified a duplication of MTM1 exon 10 both in the patient and his mother. Additional quantitative fluorescent PCR and long-range PCR revealed an additional large deletion (2536bp) within intron 10, 143bp downstream of exon 10, and confirmed the duplication of exon 10. Our findings suggest that complex rearrangements have to be considered in typically affected males with X-linked myotubular myopathy.

A.P.11

Autophagy is a highly conserved cellular degradative pathway that includes several tightly regulated steps, evolving from the initial formation of phagophores to autophagosomes, whose fusion with lysosomes results in the final structures of degradation, autolysosomes. Ectopic P-Granules Autophagy Protein 5 Homolog encoded by EPG5 is a key autophagy regulator in multicellular organisms. Recessive EPG5 mutations cause severe abnormalities of autolysosome formation and have been implicated in Vici syndrome (VS), an extensive multisystem disorder characterized by callosal agenesis, cataracts, cardiomyopathy, combined immunodeficiency and hypopigmentation. Here we report post mortem (nxa0=xa04), muscle (nxa0=xa013), liver (nxa0=xa02), nerve and skin (nxa0=xa01 each) biopsy findings from 17 individuals with VS and related EPG5-associated disorders. Muscle biopsies featured variable combinations of increased variability in fibre size with predominance of often atrophic type 1 fibres, increase in internalized nuclei, fat and connective tissue, and abnormal glycogen content. Autophagic vacuoles were conspicuous on light microscopy, often associated with basophilic stippling and acid phosphatase positive inclusions, and confirmed on electron microscopy. Other variable EM features included abnormalities of mitochondrial structure and distribution. Few patients showed additional core-like structures. Nerve biopsy findings suggested an associated neuropathy. Post mortem examination demonstrated callosal agenesis, neuromigrational abnormalities and variable hypoplasia of cerebellum, pons and corticospinal tracts as the most striking CNS abnormalities. Abnormal vacuoles and increased glycogen content were found in multiple organs, including kidney, liver and heart. These findings indicate consistent neuromuscular and CNS involvement as part of the EPG5-related phenotypical spectrum, and suggest substantial overlap with other neuromuscular conditions, and the glycogen and lysosomal storage disorders.

G.P.214

Collagen VI-related dystrophies and myopathies (COL6-RD) are a highly variable group of disorders that form a phenotypic spectrum ranging from severe Ullrich congenital muscular dystrophy (UCMD) via intermediate phenotypes to the milder Bethlem myopathy (BM). Both inter- and intra-familial variable expressivity of severity are commonly observed. We present clinical, immunohistochemical, and genetic data on four families with marked inter-generational phenotypic variability masquerading as anticipation due to parental mosaicism for a dominant mutation, with subsequent full inheritance and penetrance of the mutation in the heterozygous offspring. Additionally, we present a 5th simplex patient identified as a mosaic carrier. Parental mosaicism was confirmed in the four families through quantitative analysis of the ratio of mutant versus wild type allele in genomic DNA from various tissues; including blood, saliva, and dermal fibroblasts. Consistent with somatic mosaicism parental samples had lower ratios of mutant versus wild type allele compared to the fully heterozygote offspring, although there was notable variability of the mutant between tissues tested, ranging from 16% (saliva) to 43% (fibroblasts) in one mosaic father. This is the first report demonstrating mosaicism as a cause of intra-familial/inter-generational variability of COL6-RD, suggesting that sporadic and parental mosaicism may be more common than previously suspected.