Titin copy number variations associated with dominant inherited phenotypes.

BACKGROUND: Titinopathies are caused by mutations in the titin gene (TTN). Titin is the largest known human protein; its gene has the longest coding phase with 364 exons. Titinopathies are very complex neuromuscular pathologies due to the variable age of onset of symptoms, the great diversity of pathological and muscular impairment patterns (cardiac, skeletal muscle or mixed) and both autosomal dominant and recessive modes of transmission. Until now, only few CNVs in TTN have been reported without clear genotype-phenotype associations. METHODS: Our study includes eight families with dominant titinopathies. We performed next-generation sequencing or comparative genomic hybridisation array analyses and found CNVs in the TTN gene. We characterised these CNVs by RNA sequencing (RNAseq) analyses in six patients' muscles and performed genotype-phenotype inheritance association study by combining the clinical and biological data of these eight families. RESULTS: Seven deletion-type CNVs in the TTN gene were identified among these families. Genotype and RNAseq results showed that five deletions do not alter the reading frame and one is out-of-reading frame. The main phenotype identified was distal myopathy associated with contractures. The analysis of morphological, clinical and genetic data and imaging let us draw new genotype-phenotype associations of titinopathies. CONCLUSION: Identifying TTN CNVs will further increase diagnostic sensitivity in these complex neuromuscular pathologies. Our cohort of patients enabled us to identify new deletion-type CNVs in the TTN gene, with unexpected autosomal dominant transmission. This is valuable in establishing new genotype-phenotype associations of titinopathies, mainly distal myopathy in most of the patients.

VMA21 deficiency causes an autophagic myopathy by compromising V-ATPase activity and lysosomal acidification.

X-linked myopathy with excessive autophagy (XMEA) is a childhood-onset disease characterized by progressive vacuolation and atrophy of skeletal muscle. We show that XMEA is caused by hypomorphic alleles of the VMA21 gene, that VMA21 is the diverged human ortholog of the yeast Vma21p protein, and that like Vma21p it is an essential assembly chaperone of the V-ATPase, the principal mammalian proton pump complex. Decreased VMA21 raises lysosomal pH, which reduces lysosomal degradative ability and blocks autophagy. This reduces cellular free amino acids, which upregulates the mTOR pathway and mTOR-dependent macroautophagy, resulting in proliferation of large and ineffective autolysosomes that engulf sections of cytoplasm, merge together, and vacuolate the cell. Our results uncover macroautophagic overcompensation leading to cell vacuolation and tissue atrophy as a mechanism of disease.

ACTN2 mutations cause "Multiple structured Core Disease" (MsCD).

The identification of genes implicated in myopathies is essential for diagnosis and for revealing novel therapeutic targets. Here we characterize a novel subclass of congenital myopathy at the morphological, molecular, and functional level. Through exome sequencing, we identified de novo ACTN2 mutations, a missense and a deletion, in two unrelated patients presenting with progressive early-onset muscle weakness and respiratory involvement. Morphological and ultrastructural analyses of muscle biopsies revealed a distinctive pattern with the presence of muscle fibers containing small structured cores and jagged Z-lines. Deeper analysis of the missense mutation revealed mutant alpha-actinin-2 properly localized to the Z-line in differentiating myotubes and its level was not altered in muscle biopsy. Modelling of the disease in zebrafish and mice by exogenous expression of mutated alpha-actinin-2 recapitulated the abnormal muscle function and structure seen in the patients. Motor deficits were noted in zebrafish, and muscle force was impaired in isolated muscles from AAV-transduced mice. In both models, sarcomeric disorganization was evident, while expression of wild-type alpha-actinin-2 did not result in muscle anomalies. The murine muscles injected with mutant ACTN2 displayed cores and Z-line defects. Dominant ACTN2 mutations were previously associated with cardiomyopathies, and our data demonstrate that specific mutations in the well-known Z-line regulator alpha-actinin-2 can cause a skeletal muscle disorder.

Sarcomeric disorganization and nemaline bodies in muscle biopsies of patients with EXOSC3-related type 1 pontocerebellar hypoplasia.

INTRODUCTION: Mutations in the EXOSC3 gene are responsible for type 1 pontocerebellar hypoplasia, an autosomal recessive congenital disorder characterized by cerebellar atrophy, developmental delay, and anterior horn motor neuron degeneration. Muscle biopsies of these patients often show characteristics resembling classic spinal muscle atrophy, but to date, no distinct features have been identified. METHODS: Clinical data and muscle biopsy findings of 3 unrelated patients with EXOSC3 mutations are described. RESULTS: All patients presented as a severe congenital cognitive and neuromuscular phenotype with short survival, harboring the same point mutation (c.92G>C; p.Gly31Ala). Muscle biopsies consistently showed variable degrees of sarcomeric disorganization with myofibrillar remnants, Z-line thickening, and small nemaline bodies. CONCLUSIONS: In this uniform genetic cohort of patients with EXOSC3 mutations, sarcomeric disruption and rod structures were prominent features of muscle biopsies. In the context of neonatal hypotonia, ultrastructural studies might provide early clues for the diagnosis of EXOSC3-related pontocerebellar hypoplasia. Muscle Nerve 59:137-141, 2019.

Recessive MYPN mutations cause cap myopathy with occasional nemaline rods.

Congenital myopathies are phenotypically and genetically heterogeneous. We describe homozygous truncating mutations in MYPN in 2 unrelated families with a slowly progressive congenital cap myopathy. MYPN encodes the Z-line protein myopalladin implicated in sarcomere integrity. Functional experiments demonstrate that the mutations lead to mRNA defects and to a strong reduction in full-length protein expression. Myopalladin signals accumulate in the caps together with alpha-actinin. Dominant MYPN mutations were previously reported in cardiomyopathies. Our data uncover that mutations in MYPN cause either a cardiac or a congenital skeletal muscle disorder through different modes of inheritance. Ann Neurol 2017;81:467-473.

Affected female carriers of MTM1 mutations display a wide spectrum of clinical and pathological involvement: delineating diagnostic clues.

X-linked myotubular myopathy (XLMTM), a severe congenital myopathy, is caused by mutations in the MTM1 gene located on the X chromosome. A majority of affected males die in the early postnatal period, whereas female carriers are believed to be usually asymptomatic. Nevertheless, several affected females have been reported. To assess the phenotypic and pathological spectra of carrier females and to delineate diagnostic clues, we characterized 17 new unrelated affected females and performed a detailed comparison with previously reported cases at the clinical, muscle imaging, histological, ultrastructural and molecular levels. Taken together, the analysis of this large cohort of 43 cases highlights a wide spectrum of clinical severity ranging from severe neonatal and generalized weakness, similar to XLMTM male, to milder adult forms. Several females show a decline in respiratory function. Asymmetric weakness is a noteworthy frequent specific feature potentially correlated to an increased prevalence of highly skewed X inactivation. Asymmetry of growth was also noted. Other diagnostic clues include facial weakness, ptosis and ophthalmoplegia, skeletal and joint abnormalities, and histopathological signs that are hallmarks of centronuclear myopathy such as centralized nuclei and necklace fibers. The histopathological findings also demonstrate a general disorganization of muscle structure in addition to these specific hallmarks. Thus, MTM1 mutations in carrier females define a specific myopathy, which may be independent of the presence of an XLMTM male in the family. As several of the reported affected females carry large heterozygous MTM1 deletions not detectable by Sanger sequencing, and as milder phenotypes present as adult-onset limb-girdle myopathy, the prevalence of this myopathy is likely to be greatly underestimated. This report should aid diagnosis and thus the clinical management and genetic counseling of MTM1 carrier females. Furthermore, the clinical and pathological history of this cohort may be useful for therapeutic projects in males with XLMTM, as it illustrates the spectrum of possible evolution of the disease in patients surviving long term.

Dihydropyridine receptor (DHPR, CACNA1S) congenital myopathy.

Muscle contraction upon nerve stimulation relies on excitation-contraction coupling (ECC) to promote the rapid and generalized release of calcium within myofibers. In skeletal muscle, ECC is performed by the direct coupling of a voltage-gated L-type Ca2+ channel (dihydropyridine receptor; DHPR) located on the T-tubule with a Ca2+ release channel (ryanodine receptor; RYR1) on the sarcoplasmic reticulum (SR) component of the triad. Here, we characterize a novel class of congenital myopathy at the morphological, molecular, and functional levels. We describe a cohort of 11 patients from 7 families presenting with perinatal hypotonia, severe axial and generalized weakness. Ophthalmoplegia is present in four patients. The analysis of muscle biopsies demonstrated a characteristic intermyofibrillar network due to SR dilatation, internal nuclei, and areas of myofibrillar disorganization in some samples. Exome sequencing revealed ten recessive or dominant mutations in CACNA1S (Cav1.1), the pore-forming subunit of DHPR in skeletal muscle. Both recessive and dominant mutations correlated with a consistent phenotype, a decrease in protein level, and with a major impairment of Ca2+ release induced by depolarization in cultured myotubes. While dominant CACNA1S mutations were previously linked to malignant hyperthermia susceptibility or hypokalemic periodic paralysis, our findings strengthen the importance of DHPR for perinatal muscle function in human. These data also highlight CACNA1S and ECC as therapeutic targets for the development of treatments that may be facilitated by the previous knowledge accumulated on DHPR.

Constitutive activation of the calcium sensor STIM1 causes tubular-aggregate myopathy.

Tubular aggregates are regular arrays of membrane tubules accumulating in muscle with age. They are found as secondary features in several muscle disorders, including alcohol- and drug-induced myopathies, exercise-induced cramps, and inherited myasthenia, but also exist as a pure genetic form characterized by slowly progressive muscle weakness. We identified dominant STIM1 mutations as a genetic cause of tubular-aggregate myopathy (TAM). Stromal interaction molecule 1 (STIM1) is the main Ca(2+) sensor in the endoplasmic reticulum, and all mutations were found in the highly conserved intraluminal Ca(2+)-binding EF hands. Ca(2+) stores are refilled through a process called store-operated Ca(2+) entry (SOCE). Upon Ca(2+)-store depletion, wild-type STIM1 oligomerizes and thereby triggers extracellular Ca(2+) entry. In contrast, the missense mutations found in our four TAM-affected families induced constitutive STIM1 clustering, indicating that Ca(2+) sensing was impaired. By monitoring the calcium response of TAM myoblasts to SOCE, we found a significantly higher basal Ca(2+) level in TAM cells and a dysregulation of intracellular Ca(2+) homeostasis. Because recessive STIM1 loss-of-function mutations were associated with immunodeficiency, we conclude that the tissue-specific impact of STIM1 loss or constitutive activation is different and that a tight regulation of STIM1-dependent SOCE is fundamental for normal skeletal-muscle structure and function.

Cylindrical spirals associated with severe congenital muscle weakness and epileptic encephalopathy.

INTRODUCTION: Cylindrical spirals are characteristic muscular inclusions consisting of spiraling double-laminated membranes. They are found in heterogeneous clinical conditions. METHODS: We obtained muscle biopsies from 2 young sisters with severe congenital hypotonia, muscle weakness, and epileptic encephalopathy, and identified cylindrical spirals. RESULTS: We found an association between congenital encephalomyopathy and cylindrical spirals. CONCLUSIONS: In this morphological and ultrastructural study, we speculate on the origin of these peculiar structures.

Adult-onset autosomal dominant centronuclear myopathy due to BIN1 mutations.

Centronuclear myopathies are congenital muscle disorders characterized by type I myofibre predominance and an increased number of muscle fibres with nuclear centralization. The severe neonatal X-linked form is due to mutations in MTM1, autosomal recessive centronuclear myopathy with neonatal or childhood onset results from mutations in BIN1 (amphiphysin 2), and dominant cases were previously associated to mutations in DNM2 (dynamin 2). Our aim was to determine the genetic basis and physiopathology of patients with mild dominant centronuclear myopathy without mutations in DNM2. We hence established and characterized a homogeneous cohort of nine patients from five families with a progressive adult-onset centronuclear myopathy without facial weakness, including three sporadic cases and two families with dominant disease inheritance. All patients had similar histological and ultrastructural features involving type I fibre predominance and hypotrophy, as well as prominent nuclear centralization and clustering. We identified heterozygous BIN1 mutations in all patients and the molecular diagnosis was complemented by functional analyses. Two 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. Immunolabelling experiments revealed abnormal central accumulation of dynamin 2, caveolin-3, and the autophagic marker p62, and general membrane alterations of the triad, the sarcolemma, and the basal lamina as potential pathological mechanisms. In conclusion, we identified BIN1 as the second gene for dominant centronuclear myopathy. Our data provide the evidence that specific BIN1 mutations can cause either recessive or dominant centronuclear myopathy and that both disorders involve different pathomechanisms.

Clinical, histological and genetic characterisation of patients with tubular aggregate myopathy caused by mutations in STIM1.

BACKGROUND: Tubular aggregate myopathies (TAMs) are muscle disorders characterised by abnormal accumulations of densely packed single-walled or double-walled membrane tubules in muscle fibres. Recently, STIM1, encoding a major calcium sensor of the endoplasmic reticulum, was identified as a TAM gene. METHODS: The present study aims to define the clinical, histological and ultrastructural phenotype of tubular aggregate myopathy and to assess the STIM1 mutation spectrum. RESULTS: We describe six new TAM families harbouring one known and four novel STIM1 mutations. All identified mutations are heterozygous missense mutations affecting highly conserved amino acids in the calcium-binding EF-hand domains, demonstrating the presence of a mutation hot spot for TAM. We show that the mutations induce constitutive STIM1 clustering, strongly suggesting that calcium sensing and consequently calcium homoeostasis is impaired. Histological and ultrastructural analyses define a common picture with tubular aggregates labelled with Gomori trichrome and Nicotinamide adenine dinucleotide (NADH) tetrazolium reductase, substantiating their endoplasmic reticulum origin. The aggregates were observed in both fibre types and were often accompanied by nuclear internalisation and fibre size variability. The phenotypical spectrum ranged from childhood onset progressive muscle weakness and elevated creatine kinase levels to adult-onset myalgia without muscle weakness and normal CK levels. CONCLUSIONS: The present study expands the phenotypical spectrum of STIM1-related tubular aggregate myopathy. STIM1 should therefore be considered for patients with tubular aggregate myopathies involving either muscle weakness or myalgia as the first and predominant clinical sign.

Autosomal dominant eccentric core disease caused by a heterozygous mutation in the MYH7 gene.

BACKGROUND: Autosomal dominant (AD) central core disease (CCD) is a congenital myopathy characterised by the presence of cores in the muscle fibres which correspond to broad areas of myofibrils disorganisation, Z-line streaming and lack of mitochondria. Heterozygous mutations in the RYR1 gene were observed in the large majority of AD-CCD families; however, this gene was excluded in some of AD-CCD families. OBJECTIVE: To enlarge the genetic spectrum of AD-CCD demonstrating mutations in an additional gene. PATIENTS AND METHODS: Four affected AD family members over three generations, three of whom were alive and participate in the study: the mother and two of three siblings. The symptoms began during the early childhood with mild delayed motor development. Later they developed mainly tibialis anterior weakness, hypertrophy of calves and significant weakness (amyotrophic) of quadriceps. No cardiac or ocular involvement was noted. RESULTS: The muscle biopsies sections showed a particular pattern: eccentric cores in type 1 fibres, associated with type 1 predominance. Most cores have abrupt borders. Electron microscopy confirmed the presence of both unstructured and structured cores. Exome sequencing analysis identified a novel heterozygous missense mutation p.Leu1723Pro in MYH7 segregating with the disease and affecting a conserved residue in the myosin tail domain. CONCLUSIONS: We describe MYH7 as an additional causative gene for AD-CCD. These findings have important implications for diagnosis and future investigations of AD-congenital myopathies with cores, without cardiomyopathy, but presenting a particular involvement of distal and quadriceps muscles.

Mutations in dynamin 2 cause dominant centronuclear myopathy.

Autosomal dominant centronuclear myopathy is a rare congenital myopathy characterized by delayed motor milestones and muscular weakness. In 11 families affected by centronuclear myopathy, we identified recurrent and de novo missense mutations in the gene dynamin 2 (DNM2, 19p13.2), which encodes a protein involved in endocytosis and membrane trafficking, actin assembly and centrosome cohesion. The transfected mutants showed reduced labeling in the centrosome, suggesting that DNM2 mutations might cause centronuclear myopathy by interfering with centrosome function.

[Congenital myasthenic syndromes; French experience]. / Syndromes myasthéniques congénitaux - L'expérience française.

Congenital myasthenic syndromes CMS) form a heterogeneous group of genetic diseases characterized by abnormal neuromuscular transmission. The associated muscular weakness is exacerbated by exertion and usually starts during infancy/childhood In 2002 a national Congenital Myasthenic Syndromes Network was created in France, composed of neurologists, neuropediatricians, pathologists, molecular geneticists and neurobiologists. The network has now identified nearly 300 cases of CMS, as well as three new culprit genes. Based on our personal experience and data from the most recent studies, we describe the 18 principal culprit genes so far identified, along with diagnostic pitfalls, the disease course, prognosis and treatment. The underlying genetic defect remains to be identified in nearly half of CMS patients.

VMA21 deficiency prevents vacuolar ATPase assembly and causes autophagic vacuolar myopathy.

X-linked Myopathy with Excessive Autophagy (XMEA) is a childhood onset disease characterized by progressive vacuolation and atrophy of skeletal muscle. We show that XMEA is caused by hypomorphic alleles of the VMA21 gene, that VMA21 is the diverged human ortholog of the yeast Vma21p protein, and that like Vma21p, VMA21 is an essential assembly chaperone of the vacuolar ATPase (V-ATPase), the principal mammalian proton pump complex. Decreased VMA21 raises lysosomal pH which reduces lysosomal degradative ability and blocks autophagy. This reduces cellular free amino acids which leads to downregulation of the mTORC1 pathway, and consequent increased macroautophagy resulting in proliferation of large and ineffective autolysosomes that engulf sections of cytoplasm, merge, and vacuolate the cell. Our results uncover a novel mechanism of disease, namely macroautophagic overcompensation leading to cell vacuolation and tissue atrophy.

Identification of an agrin mutation that causes congenital myasthenia and affects synapse function.

We report the case of a congenital myasthenic syndrome due to a mutation in AGRN, the gene encoding agrin, an extracellular matrix molecule released by the nerve and critical for formation of the neuromuscular junction. Gene analysis identified a homozygous missense mutation, c.5125G>C, leading to the p.Gly1709Arg variant. The muscle-biopsy specimen showed a major disorganization of the neuromuscular junction, including changes in the nerve-terminal cytoskeleton and fragmentation of the synaptic gutters. Experiments performed in nonmuscle cells or in cultured C2C12 myotubes and using recombinant mini-agrin for the mutated and the wild-type forms showed that the mutated form did not impair the activation of MuSK or change the total number of induced acetylcholine receptor aggregates. A solid-phase assay using the dystrophin glycoprotein complex showed that the mutation did not affect the binding of agrin to alpha-dystroglycan. Injection of wild-type or mutated agrin into rat soleus muscle induced the formation of nonsynaptic acetylcholine receptor clusters, but the mutant protein specifically destabilized the endogenous neuromuscular junctions. Importantly, the changes observed in rat muscle injected with mutant agrin recapitulated the pre- and post-synaptic modifications observed in the patient. These results indicate that the mutation does not interfere with the ability of agrin to induce postsynaptic structures but that it dramatically perturbs the maintenance of the neuromuscular junction.

COL4A1 mutations and hereditary angiopathy, nephropathy, aneurysms, and muscle cramps.

BACKGROUND: COL4A3, COL4A4, and COL4A5 are the only collagen genes that have been implicated in inherited nephropathies in humans. However, the causative genes for a number of hereditary multicystic kidney diseases, myopathies with cramps, and heritable intracranial aneurysms remain unknown. METHODS: We characterized the renal and extrarenal phenotypes of subjects from three families who had an autosomal dominant hereditary angiopathy with nephropathy, aneurysms, and muscle cramps (HANAC), which we propose is a syndrome. Linkage studies involving microsatellite markers flanking the COL4A1-COL4A2 locus were performed, followed by sequence analysis of COL4A1 complementary DNA extracted from skin-fibroblast specimens from the subjects. RESULTS: We identified three closely located glycine mutations in exons 24 and 25 of the gene COL4A1, which encodes procollagen type IV alpha1. The clinical renal manifestations of the HANAC syndrome in these families include hematuria and bilateral, large cysts. Histologic analysis revealed complex basement-membrane defects in kidney and skin. The systemic angiopathy of the HANAC syndrome appears to affect both small vessels and large arteries. CONCLUSIONS: COL4A1 may be a candidate gene in unexplained familial syndromes with autosomal dominant hematuria, cystic kidney disease, intracranial aneurysms, and muscle cramps.

The clinical, histologic, and genotypic spectrum of SEPN1-related myopathy: A case series.

OBJECTIVE: To clarify the prevalence, long-term natural history, and severity determinants of SEPN1-related myopathy (SEPN1-RM), we analyzed a large international case series. METHODS: Retrospective clinical, histologic, and genetic analysis of 132 pediatric and adult patients (2-58 years) followed up for several decades. RESULTS: The clinical phenotype was marked by severe axial muscle weakness, spinal rigidity, and scoliosis (86.1%, from 8.9 ± 4 years), with relatively preserved limb strength and previously unreported ophthalmoparesis in severe cases. All patients developed respiratory failure (from 10.1±6 years), 81.7% requiring ventilation while ambulant. Histopathologically, 79 muscle biopsies showed large variability, partly determined by site of biopsy and age. Multi-minicores were the most common lesion (59.5%), often associated with mild dystrophic features and occasionally with eosinophilic inclusions. Identification of 65 SEPN1 mutations, including 32 novel ones and the first pathogenic copy number variation, unveiled exon 1 as the main mutational hotspot and revealed the first genotype-phenotype correlations, bi-allelic null mutations being significantly associated with disease severity (p = 0.017). SEPN1-RM was more severe and progressive than previously thought, leading to loss of ambulation in 10% of cases, systematic functional decline from the end of the third decade, and reduced lifespan even in mild cases. The main prognosis determinants were scoliosis/respiratory management, SEPN1 mutations, and body mass abnormalities, which correlated with disease severity. We propose a set of severity criteria, provide quantitative data for outcome identification, and establish a need for age stratification. CONCLUSION: Our results inform clinical practice, improving diagnosis and management, and represent a major breakthrough for clinical trial readiness in this not so rare disease.

"Necklace" fibers, a new histological marker of late-onset MTM1-related centronuclear myopathy.

Mutations in the gene encoding the phosphoinositide phosphatase myotubularin 1 protein (MTM1) are usually associated with severe neonatal X-linked myotubular myopathy (XLMTM). However, mutations in MTM1 have also been recognized as the underlying cause of "atypical" forms of XLMTM in newborn boys, female infants, female manifesting carriers and adult men. We reviewed systematically the biopsies of a cohort of patients with an unclassified form of centronuclear myopathy (CNM) and identified four patients presenting a peculiar histological alteration in some muscle fibers that resembled a necklace ("necklace fibers"). We analyzed further the clinical and morphological features and performed a screening of the genes involved in CNM. Muscle biopsies in all four patients demonstrated 4-20% of fibers with internalized nuclei aligned in a basophilic ring (necklace) at 3 microm beneath the sarcolemma. Ultrastructurally, such necklaces consisted of myofibrils of smaller diameter, in oblique orientation, surrounded by mitochondria, sarcoplasmic reticulum and glycogen granules. In the four patients (three women and one man), myopathy developed in early childhood but was slowly progressive. All had mutations in the MTM1 gene. Two mutations have previously been reported (p.E404K and p.R241Q), while two are novel; a c.205_206delinsAACT frameshift change in exon 4 and a c.1234A>G mutation in exon 11 leading to an abnormal splicing and the deletion of nine amino acids in the catalytic domain of MTM1. Necklace fibers were seen neither in DNM2- or BIN1-related CNM nor in males with classical XLMTM. The presence of necklace fibers is useful as a marker to direct genetic analysis to MTM1 in CNM.