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TorsinA-interacting protein 1 (TOR1AIP1) gene is a novel gene that has recently been described to cause limb-girdle muscular dystrophy (LGMD) with mild dilated cardiomyopathy. We report a family with mutations in TOR1AIP1 where the striking clinical feature is severe cardiac failure requiring cardiac transplant in two siblings, in addition to musculoskeletal weakness and muscular dystrophy. We demonstrate an absence of TOR1AIP1 protein expression in cardiac and skeletal muscles of affected siblings. We expand the phenotype of this gene to demonstrate the cardiac involvement and the importance of cardiac surveillance in patients with mutations in TOR1AIP1.

Cap disease or cap myopathy is a form of congenital myopathy in which peripheral, well-demarcated 'caps' of disorganised thin filaments are seen in muscle fibres. Mutation of the TPM2 gene, that encodes beta-tropomyosin, is the first reported genetic cause. In this paper, we describe a further case of cap disease due to a mutation in TPM2, confirming the importance of this genetic association. This is the first report of cardiac dysfunction due to a mutation in TPM2. Our patient has an identical TPM2 mutation to the first genetically diagnosed cap disease patient, a denovo heterozygous three base pair deletion that removes glutamic acid 139 from the centre of beta-tropomyosin (p.E139del). 2D-gel electrophoresis studies show that the shortened mutant protein incorporates into sarcomeric structures, where it likely imposes a dominant-negative effect to cause muscle weakness.

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.

Life-threatening cardiac and respiratory complications are common in LMNA-related myopathies and early diagnosis is important for optimal patient care. Lamin A/C related congenital muscular dystrophy (L-CMD) is often caused by de novo mutation in LMNA, affecting a single child in a family. Germinal mosaicism is a rarer variant that can lead to two children inheriting the same new heterozygous mutation from a clinically unaffected parent. Both patterns mimic autosomal recessive (AR) inheritance and the possibility of de novo L-CMD may be forgotten since most causes of congenital muscular dystrophy follow AR inheritance. To illustrate the challenge of diagnosing L-CMD, we present a consanguineous family in which two children have early onset LMNA-related myopathy likely due to paternal germinal mosaicism. This emphasises that germinal mosaicism (and de novo mutations) for LMNA can arise in any family and direct gene sequencing is required to confirm or exclude the diagnosis.

Nemaline myopathy (NM) is a disorder of the skeletal muscle thin filament characterised by muscle dysfunction and electron-dense protein accumulations (nemaline bodies). Pathogenic mutations have been described in nine genes to date, but the genetic basis remains unknown in many cases. We used whole exome sequencing (WES) in two families with NM and subsequent gene sequencing in over 540 additional genetically unresolved NM patients to identify and characterise a new genetic cause of NM. We developed a knock-down zebrafish model of this condition and used immunohistochemistry, western blotting, single-fibre contractility studies and recombinant protein studies to characterise the expression, localisation and biochemical functions of the new disease-related protein. We identified homozygous or compound heterozygous variants in LMOD3, which encodes leiomodin-3 (Lmod3) in 21 patients from 14 families. Affected individuals had severe generalised weakness and hypotonia, and most affected individuals died in the neonatal period. We demonstrated that Lmod3 is expressed from early muscle differentiation, localises to thin filaments with enrichment at the pointed ends, and has strong actin nucleating activity. Loss of Lmod3 in patient muscle results in shortening and disorganisation of thin filaments. Knockdown of lmod3 in the zebrafish replicates this phenotype. These findings define a new genetic subtype of congenital myopathy and demonstrate an essential, previously unrecognised role for Lmod3 in the regulation of sarcomeric thin filaments in skeletal muscle.

Tropomyosin 3 encoded by the TPM3 gene is a member of the acting binding tropomyosin family, a component of the sarcomeric thin filaments troponin tropomyosin complex that is essential in muscle contraction by regulating the calcium dependent binding of the myosin head to the actin filament in anticipation of the force generating power stroke. Mutations in TPM3 cause a clinical and histopathological heterogeneous group of neuromuscular disorders characterized by progressive weakness that includes CAP myopathy, congenital fiber type disproportion and nemaline myopathy. Recent studies of the deltaK7 mutation in TPM2 suggest that the pathogenic mechanism underlying tropomyosin related disease with progressive muscle contractures may be a gain of function change, leading to elevated Ca 2+ sensitivity of force generation, while this has not yet been shown conclusively for TPM3 . Here we report two unrelated patients with a de novo TPM3 single glutamic acid deletions resulting in a much more significant contractile phenotype with marked congenital muscle stiffness associated with ventilator failure in one case. We hypothesize that these single amino acid deletions result in increased Ca 2+ sensitivity of the troponin tropomyosin complex and a consequently hypercontractile sarcomere. We thus expand the clinical, phenotypic and pathophysiological spectrum of TPM3 mutations, which now join recessive α B-crystallin mutations and specific ACTA1 and TPM2 mutations as a myogenic cause for neonatal muscle rigidity.

Limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of genetic disorders associated with proximal muscle weakness that begins after 2 years of age and dystrophic changes on muscle biopsy. The genetic cause of LGMD in Australia remains uncertain in approximately 40% of patients resulting in uncertainty about reproductive risks for patients and long term prognosis. We reviewed the clinical information in a large cohort of patients with LGMD who lacked a genetic diagnosis, usually after extensive previous investigations ( n = 50). We first screened patients for myotonic dystrophy type 2 and then performed whole exome sequencing (WES), analysing data using the ‘xBrowse’ web interface (Broad Institute). Testing for myotonic dystrophy type 2 (DM2) identified 2 families in our LGMD cohort. Using WES we identified likely pathogenic mutations in known myopathy genes in 20 families, often in genes not typically associated with LGMD. Two families were diagnosed with dominant LGMD, 9 families with recessive LGMD, 3 with mutations congenital muscular dystrophy genes, 3 with collagen myopathies, 2 with metabolic myopathies and 1 in ACTA1. In addition one family had a mutation CHD7, a gene usually associated with CHARGE syndrome. Our overall diagnostic success rate was 46% in our LGMD cohort of patients most of whom had been extensively investigated previously and who remained without a genetic diagnosis. The diversity of genetic causes that we identified supports the clinical observation of that LGMD is highly heterogeneous and shares clinical features with many other forms of myopathy. Our study emphasises the challenges that clinicians face making a genetic diagnosis in all families. Whole exome sequencing and similar next-generation sequencing are likely to be efficient and cost-effective methods for identifying the genetic basis of LGMD but clinicians must remember that some disorders in the differential diagnosis, such as DM2, are routinely missed by WES.

Small heat-shock protein-beta 8 (HSPB8) belongs to the superfamily of small heat-shock proteins that act as stress proteins with chaperon-like activity in the cells. Missense mutations in a gene encoding HSPB8 protein have been associated with neuromuscular diseases: distal hereditary motor neuropathy 2A (dHMN2A) and Charcot–Marie–Tooth disease (CMT2L). Mutations located in the α-crystallin domain of HspB8 cause decreased chaperone-like activity and incomplete clearance of aggregated proteins. We identified the same mutation (c.421A > G p.K141E) that has earlier been associated to dHMN2A and CMT2L diseases in a family clinically classified as a distal myopathy. In addition, we identified a new mutation, c.515insC p.P173SfsX43, in another distal myopathy family. Thorough clinical, electrophysiology, imaging and pathology examinations show that the HSPB8 mutations in these families lead to both motor neuropathic as well as myopathic changes accounting for the distal presentation. The pathological changes in the muscle were characterized by both neurogenic findings and myofibrillar myopathy with autophagic rimmed vacuoles. The extensive immune histochemical and immunofluorescence muscle pathology studies in this chaperonopathy will be presented together with muscle imaging.

Duchenne muscular dystrophy (DMD) is a progressive disorder that affects 1 in 3500 males and that typically results in death between 20 and 35 years of age from respiratory or cardiac failure. International standards of care have been devised to guide the management of DMD patients. Due to its high incidence in all populations of the world and relatively homogeneous natural history, health outcomes in DMD provide a useful yardstick by which to assess neuromuscular services between regions. There is currently little data available on how males with DMD in NSW, Australia, fare in terms of health outcomes. Such data would provide health service providers important feedback about the effectiveness of neuromuscular services in NSW compared to other populations in Australia and overseas. This study was designed to conduct an audit of all paediatric and adult neuromuscular services in NSW to document the morbidity and mortality of patients with Duchenne muscular dystrophy in NSW in the last 10 years, and evaluate their transition experience to adult services. In addition we describe the current range of paediatric and adult neuromuscular services available in NSW and review the literature to identify and evaluate the effectiveness of different transition models that have been developed internationally and within Australia.

The dystrophinopathies are allelic muscular dystrophies caused by X-linked recessive mutations in dystrophin, with only rare reports of asymptomatic adult males. The static cognitive impairment seen in dystrophinopathies is thought to be due to altered expression of dystrophin isoforms, but has only once been described in the absence of muscle weakness. We identified a cohort of patients with unexpected copy number variants (CNV) in the dystrophin gene, on microarrays performed for developmental delay or intellectual disability, in whom muscle weakness was minimal or absent. Subjects with a dystrophin CNV referred to the neurology or genetics departments at RCH Melbourne or GHSV were assessed. An additional family was identified from the CHW, and included. Twelve probands had a CNV in the dystrophin gene on microarray testing. Eight (seven male, one female; seven deletions, one duplication), age 0–9 years, had atypical phenotypes as described above. CNVs were found in 10 family members (five males and five females), including three asymptomatic adult males. In all but one family, MLPA confirmed loss of exons. Muscle weakness was absent or minimal. Serum CK was normal or mildly elevated. Muscle biopsy revealed morphologically normal muscle with normal dystrophin immunoreactivity. Microarray testing has revealed an extended spectrum of clinical phenotypes associated with mutations in dystrophin, that may include isolated developmental delay and asymptomatic individuals. Further study is required to understand the molecular basis of the apparent absence of muscle pathology in these patients, and the relationship of the dystrophin deletion to cognitive impairment.

The limb-girdle MDs (LGMDs) are characterised by predominant weakness and wasting of pelvic and shoulder girdle muscles with onset after two years of age. Recessive mutations in the TCAP gene (encoding telethonin), were first identified as the cause of LGMD2G in 2000. Only a handful of families have been reported to date. Here, we report the ninth family with LGMD2G. The proband (P1) is a 32 year old male with an affected sister, five unaffected siblings and first-cousin parents of Chinese-Cambodian descent. P1 had slowly progressive muscle weakness since his late teenage years. On examination at 27 years of age, he had bilateral Achilles contractures, mild calf hypertrophy, asymmetric scapular winging and normal facial strength except mild weakness of eye closure. There was moderate weakness of hip movements and knee extension and mild weakness of most other muscle groups, except hand and wrist movements. We performed genome-wide linkage analysis assuming autosomal recessive inheritance using Illumina 660WQ SNP platform and found positive LOD scores for the chromosomal region 17p11.2-q22, containing the TCAP gene, as well as regions on chromosomes 7, 16 and 18. Sanger sequencing of TCAP revealed a homozygous 8 base pair duplication (c.26_33dupAGGTGTCG) resulting in a frameshift and truncated protein (p.R12fsX31), predicted to abolish telethonin expression. Two other families of Chinese origin have been reported with the same homozygous mutation. A muscle MRI at 28 years of age demonstrated widespread asymmetric fatty infiltration of lower limb muscles in a pattern similar to that reported in another LGMD2G patient by CT imaging. In conclusion, LGMD2G is rare, but exists outside Brazil and our identification of a third Asian LGMD2G family with the p.R12fsX31 mutation suggests this may be a population-specific mutation. Consistency in the pattern of muscle involvement on MRI or CT in two LGMD2G patients suggests this investigation may be a useful tool for diagnosis.

TTN encodes titin, the largest human protein. In striated muscle two titin molecules align “head to head” to span the full length of the sarcomere, providing a scaffold for sarcomere organisation, a sensing and signalling hub, and both passive and active modulation of muscle contraction. Dominant mutations in TTN are established causes of cardiomyopathy, tibial muscular dystrophy, and hereditary myopathy with early respiratory failure. More recently, several individuals with two truncating mutations have been described with increased internalised nuclei (CNM) with or without multi-minicores (MMC). Using next generation sequencing, we identified 14 individuals from 11 families with compound heterozygous or homozygous truncating TTN mutations. Presentation was in utero or during infancy in all cases. Weakness of truncal and respiratory muscles was often prominent, and early-onset scoliosis and respiratory failure were common complications. Three affected individuals had dilated cardiomyopathy (DCM) or left ventricular dysfunction, and one carrier parent developed DCM in later life. Two individuals had congenital aortic abnormalities (coarctation and stenosis). Many affected individuals had distinctive clinical features which are unusual for congenital myopathies, including congenital or early-onset hand and foot deformities, congenital scoliosis, spinal rigidity, cleft palate, distal hypermobility and short stature. The most common histological abnormality was a mixed CNM-MMC pattern. Novel histological patterns included typical congenital fibre-type disproportion (CFTD) and CNM-MMC with caps and nemaline rods. The presence of palatal clefts, facial dysmorphology, and cardiac malformations in several individuals with TTN mutations suggests that titin plays a role in foetal development. Mutations in TTN should be considered in any individual with predominant truncal and respiratory weakness, and CNM, MMC, CFTD, caps, rods, or any combination of these, on muscle biopsy.

Myosin myopathies comprise a group of inherited diseases caused by mutations in myosin heavy chain (MyHC) genes. Homozygous or compound heterozygous truncating MYH2 mutations have been demonstrated to cause recessive myopathy with ophthalmoplegia, mild to moderate muscle weakness and complete lack of type 2A muscle fibers. In this study, we describe the clinical and morphological characteristics of recessive MYH2 missense mutations. Seven patients of five different families with a myopathy characterized by ophthalmoplegia and mild to moderate muscle weakness were investigated. Muscle biopsy was performed to study morphological changes and MyHC isoform expression. Five of the patients were homozygous for MYH2 missense mutations, one patient was compound heterozygous for a missense and a nonsense mutation and one patient was homozygous for a frame-shift MYH2 mutation. Muscle biopsy demonstrated morphologically abnormal or absent type 2A muscle fibers and reduced or absent expression of the corresponding MyHC IIa transcript and protein. We conclude that missense mutations in MYH2 may cause a recessively inherited myopathy associated with external ophthalmoplegia, similar to that of truncating MYH2 mutations, but with different type 2 fiber pathology.

Background: RYR1 mutations are typically associated with core myopathies and are the most common overall cause of congenital myopathy. Dominant mutations are most often associated with central core disease and malignant hyperthermia, and genotype-phenotype patterns have emerged from the study of these mutations that have contributed to the understanding of disease pathogenesis. The recent availability of genetic testing for the entire RYR1 coding sequence has led to a dramatic expansion in the identification of recessive mutations in core myopathies and other congenital myopathies. To date, no clear patterns have been identified in these recessive mutations, though no systematic examination has yet been performed. Methods: In this study, we investigated genotype-phenotype correlations in a large combined cohort of unpublished (n = 14) and previously reported (n = 92) recessive RYR1 cases. Results: Overall examination of this cohort revealed nearly 50% of cases to be non-core myopathy related. Our most significant finding was that hypomorphic mutations (mutations expected to diminish RyR1 expression) were enriched in patients with severe clinical phenotypes. We also determined that hypomorphic mutations were more likely to be encountered in non-central core myopathies. With analysis of the location of non-hypomorphic mutations, we found that missense mutations were generally enriched in the MH/CCD hotspots and specifically enriched in the selectivity filter of the channel pore. Conclusions: These results support a hypothesis that loss of protein function is a key predictive disease parameter. In addition, they suggest that decreased RyR1 expression may dictate non-core related pathology though, data on protein expression was limited and should be confirmed in a larger cohort. Lastly, the results implicate abnormal ion conductance through the channel pore in the pathogenesis in recessive core myopathies. Overall, our findings represent a comprehensive analysis of genotype-phenotype associations in recessive RYR1-myopathies.

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.

In patients with repeated episodes of rhabdomyolysis, a predisposing condition should be suspected. Most inherited causes are related to metabolic myopathies and malignant hyperthermia susceptibility. However in the majority of patients with recurrent rhabdomyolysis a cause is not identified. To genetically and clinically characterize a cohort of patients with recurrent rhabdomyolysis we applied next generation sequencing methods to study a panel of 277 genes, mutations in which have been associated with neuromuscular diseases, plus Sanger sequencing of not well covered target genomic regions. Twenty-two patients were included in the study. Two were excluded for lack of clinical data available. None of them had affected relatives. Of the remaining patients six were female. Average peak CK was 61,000. Basal CK was mildly elevated in 9 and moderately in 1. No patient had fixed weakness. Seven had symptoms between episodes, predominantly aches and cramps. Rhabdomyolysis episodes were spontaneous in 5 patients. The most common trigger was exercise. Muscle biopsies showed type 2 fibre predominance in 4, cores in 3 and increased cytoplasmic lipid and glycogen in 1. Two patients had variants in RYR1, 3 in CACNA1S, 2 in SCN4A, 1 in PHKA1 and 2 were compound heterozygous for variants in HSPG2 (perlecan). Except for one of the RYR1 variants and one in HSPG2 all the rest are novel variants, with pathogenicity yet to be confirmed. In conclusion, only one of our patients had a definite diagnosis. Some other candidate genes harbour possibly pathogenic mutations, most of them in malignant hyperthermia related genes, of which SCN4A and CACNA1s have not yet been described in association with rhabdomyolysis out of the context of malignant hyperthermia. Most of the patients in the cohort remain unresolved raising the idea that rhabdomyloysis may not result from genetic susceptibility, or the responsible genes remain to be identified.

Exome sequencing has proven to be a powerful and cost-effective approach for the identification of causal mutations in many patients suffering from rare, severe Mendelian diseases. However, exome analysis unambiguously identifies a causal mutation in only 30–50% of sequenced families, indicating much work remains to be done to increase the yield of causal variants from sequencing-based approaches. Causal mutations can be missed by current exome sequencing approaches for a variety of reasons. Conversely, there may be multiple gene candidates that require further information to prioritize for functional studies. We describe the development of an integrated pipeline for the identification of causal variants from exome data and its application to a cohort of severe, undiagnosed muscle disease patients. Our online application called xBrowse enables the intuitive analysis of family-based exome data, permitting researchers and clinicians to rapidly explore the effects of altering inheritance modes and function/quality filters on the identification of potential causal mutations. Through xBrowse, our collaborators have early access to gene-based RNA expression data across various human tissues, a disease gene-centric protein–protein interaction networks and a large reference panel of over 50,000 exomes. We have applied this integrated approach to exome data over 250 individuals consisting of families and probands affected by a range of neuromuscular diseases. We describe the detection of novel sequence variants with strong evidence for causality in these patients, and provide case studies indicating the value of tissue expression data, protein–protein interaction networks, large reference panels for the prioritization of disease-associated mutations.

Muscle disease shows extensive genetic and allelic heterogeneity, with many genes causing related phenotypes. Some of the gene loci causing muscular dystrophy are the largest in the human genome (e.g. titin, dystrophin) making molecular diagnostics particularly challenging and expensive. Next generation sequencing promises single-test approach to diagnostics. A commonly utilized nextgen approach is whole exome sequence (WES), where hybrid capture of exons enables parallel sequencing of most exons in a single sequencing run. However, WES has an exonic drop-out rate of about 10% – 1 in 10 dystrophin, nebulin, titin or other gene exons would remain ‘non-sequenced’ with the WES approach. Thus, a negative result on WES does not necessarily rule out common genetic causes of muscle disease. An alternative approach is targeted sequencing panels. Here, each exon of specific candidate myopathy genes is individually amplified by microbubble PCR (RainDance), and this highly enriched DNA is then subjected to nextgen sequencing. Here, we develop and test a targeted muscle disease gene panel for 45 of the most common myopathy genes (1851 amplimers). We report our experience with this panel in 89 myopathy patients. We found greater consistency in depth of coverage (average 1000-fold coverage), less drop-out (2% instead of 10% in exomes), and genotype/phenotype concordance with clinician-determined differential diagnosis. Overall, targeted re-sequencing panels allow for more definitive testing of known muscular dystrophy genes. We give some specific case examples, with mutation in titin (TTN), ryanodine receptor (RYR1), and nebulin (NEB) genes. Both WES and targeted sequencing panels permit more centralized testing at greatly reduced cost and more rapid turn-around compared to single gene testing. The routine implementation of nextgen sequencing approaches should streamline molecular diagnostics, genetic counseling, and patient navigation for clinical trials.

DCSMA is an autosomal dominant disorder of developing anterior horn cells, which results in lower limb predominant weakness, and wasting. HSP is a later-onset disorder of corticospinal motor neurons, which is also lower limb predominant. In a collaboration established through the Genome Variant Database for Human Diseases, we used a combination of genome-wide SNP-based linkage analysis, whole exome sequencing, and comparison of clinical and MRI features with those of other genetically characterized forms of DCSMA, to identify four mutations in a new disease gene in six kindreds with DCSMA or HSP, or a DCSMA/HSP overlap syndrome. Several novel pathogenic-looking variants in additional kindreds are also being evaluated, suggesting this gene may be a common cause of motor neuron pathology. The causative gene encodes a dynein/dynactin trafficking adaptor protein, and appears to play a key role in the trafficking of cellular cargos critical to motor neuron development and/or maintenance. Functional studies suggest that missense mutations affecting two distinct binding regions increase the protein’s affinity for the dynein/dynactin complex, which is likely to alter the specificity and/or efficiency of dynein/dynactin-mediated trafficking. In addition, localization of secretory vesicles was altered in cells transfected with the mutant protein. We postulate that perturbation of secretory vesicle trafficking results in impaired development and maintenance of a key subset of lumbar and cervical anterior horn motor neurons, and corticospinal motor neurons, leading to the anterior horn and/or upper motor neuron pathology that characterizes these disorders. This discovery underscores the importance of key elements of the highly conserved dynein/dynactin trafficking pathway in the development and survival of both lower and upper motor neurons, and further-highlights this pathway as a potentially valuable target for therapeutic intervention in motor neuron diseases.

Mutations in RYR1 are responsible for malignant hyperthermia (MH) and several forms of congenital myopathy. Dominant mutations that give rise to central core disease (CCD) and MH cluster in three hotspot regions and the disease mechanisms associated with these diseases are well studied. In comparison, little is known about how recessive mutations, which occur throughout the gene, lead to disease. To address this issue, we reviewed the literature to map potentially functional domains within the ryanodine receptor type 1 (RyR1) and then analysed the nature and position of RYR1 mutations in 74 individuals with recessive disease, together with their clinical and histological phenotypes. Hypomorphic mutations, which abolish or markedly reduce RyR1 protein expression, were associated with more severe clinical phenotypes and with histological phenotypes other than CCD, such as multi-minicore disease, centronuclear myopathy and congenital fibre type disproportion. For non-hypomorphic mutations, CCD mutations were enriched in MH/CCD hotspot 3 and non-CCD core mutations in hotspot 2. Clustering of non-hypomorphic mutations in the triadin-binding domain (which lies within hotspot region 3) highlights abnormal triadin-binding as a potentially important disease mechanism. Non-hypomorphic mutations associated with ophthalmoplegia clustered in regions involved in ‘interdomain interactions’ which play roles in stabilising the closed conformation of the RyR1 channel. These results suggest that the variability seen in recessive RYR1 histological phenotypes may stem from differences in disease mechanism, determined in part by the location of non-hypomorphic mutations. Reduced overall RyR1 function, reduced stability of the closed channel conformation and abnormal triadin-RyR1 interactions are highlighted as potentially important disease mechanisms in various recessive RYR1-myopathies.

LGMD is predominantly inherited in an autosomal recessive manner; however autosomal dominant and X-linked subtypes have also been described. To date, most mutations that have been reported in LGMD patients have been point mutations, and these lie in approximately 17 genes. There are occasional reports of deletions in autosomal recessive LGMD genes, often only discovered when a single point mutation has been found in a gene known to cause autosomal recessive LGMD. We sought to determine whether deletions and duplications are a common cause of LGMD that have been routinely missed by standard sequencing approaches. We designed a bespoke Roche Nimblegen 12x135K CGH array that allowed for the simultaneous targeted interrogation of all coding exons of 17 LGMD genes, with additional widely-spaced backbone probes providing coverage across the whole genome. Dosage changes were confirmed by real-time quantitative PCR. We used DNA from 50 undiagnosed LGMD patients ascertained at Auckland City Hospital and 100 undiagnosed patients from the INMR laboratory, Children’s Hospital at Westmead with congenital myopathy and LGMD who had been screened for most of the common genetic causes. We found copy number variations in a handful of patients and are verifying these with real-time quantitative PCR. The data suggest a low incidence of deletion and duplication mutations. In conclusion, large deletions and duplications are uncommon in the known LGMD genes in undiagnosed LGMD patients and screening for deletions and duplications is unlikely to be a useful approach, unless single recessive mutations are found or standard gene sequencing protocols fail to identify the causative mutation. Sequencing e.g. by Next Generation Sequencing methods is likely to play a significant role in ascertaining a genetic diagnosis for these patients in an affordable and timely manner.

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.

Two mutations, skeletal α-actin K326N and β-tropomyosin ΔK7 were reported in patients with ‘stiff’ muscles and spontaneous contractures, respectively, suggesting a hypercontractile phenotype. K326N actin was isolated from a patient biopsy and ΔK7 β-tropomyosin was expressed in baculovirus/sf9 cells. Ca2+ regulation of reconstituted thin filaments was studied by quantitative in vitro motility assay. Both mutations increased Ca2+-sensitivity (EC50 mut/WT = 0.37 ± 0.05 and 0.45 ± 0.25 respectively). In recent models of actin-α-tropomyosin in the OFF state, both actin K326 and tropomyosin K7 are located in the actin-tropomyosin interface. We hypothesise that the charge change due to the mutation destabilises the OFF state and moves the thin filament equilibrium towards the ON state, thus accounting for the higher Ca2+-sensitivity. We also examined a pair of mutations that cause loss of function. Skeletal α-actin D292V (from a patient biopsy) and β-tropomyosin E117K (expressed in baculovirus/sf9 cells) cause congenital fibre type disproportion (hypotonia and weak contractility with small type 1 muscle fibres but without nemaline bodies). In reconstituted thin filaments β-tropomyosin E117K caused a decrease in Ca2+-sensitivity (EC50 mut/WT = 2.44 ± 0.55). Addition of tropomyosin to actin D292V filaments caused a complete switch-OFF of motility that could not be reversed by troponin at high Ca2+ or even NEM-S-1. Thus congenitally weak muscles correlate with loss of function at the molecular level. These mutations are not at the interface of the OFF state but have an opposite charge change to the gain-of-function mutations and are in a location that could destabilise the ON state, which may account for the loss of function.

later age of onset for dysferlinopathy patients with non-truncating mutations. We analyzed the clinical history of a large cohort of dysferlinopathy patients from both the Jain Foundation registry and the published literature to determine the effect of gender and type of mutation on the age of onset. The total cohort studied includes 321 dysferlinopathy patients with confirmed mutations and 420 diagnosed at either a genetic or protein level. In our patient cohort, 77% of patients have onset between 15 and 30 years of age, with about 12% each having onset before age 15 or after age 30. The average age of onset is similar between females (21.9 years) and males (21.3 years). However, females are more likely to have onset in early adolescence (i.e. 33% of females vs. 17% of males have onset by age 16) and after age 40. Conversely, 45% ofmale patients, but only 25%of females, have onset in the age range of 17–20. These observations suggest that the onset of symptoms may be related to some aspect of maturation. We also found a small, but noticeable effect of mutation type. Althoughpatientswith either truncating or non-truncatingmutations can have early or late onset, a statistically-significant majority of patients with onset after age 35 have at least one non-truncating mutation. In conclusion, our data indicate that both gender and type of mutation influence the age of onset in dysferlinopathy.