Edoardo Malfatti

Infantile Encephalopathy and Defective Mitochondrial DNA Translation in Patients with Mutations of Mitochondrial Elongation Factors EFG1 and EFTu

Mitochondrial protein translation is a complex process performed within mitochondria by an apparatus composed of mitochondrial DNA (mtDNA)-encoded RNAs and nuclear DNA-encoded proteins. Although the latter by far outnumber the former, the vast majority of mitochondrial translation defects in humans have been associated with mutations in RNA-encoding mtDNA genes, whereas mutations in protein-encoding nuclear genes have been identified in a handful of cases. Genetic investigation involving patients with defective mitochondrial translation led us to the discovery of novel mutations in the mitochondrial elongation factor G1 (EFG1) in one affected baby and, for the first time, in the mitochondrial elongation factor Tu (EFTu) in another one. Both patients were affected by severe lactic acidosis and rapidly progressive, fatal encephalopathy. The EFG1-mutant patient had early-onset Leigh syndrome, whereas the EFTu-mutant patient had severe infantile macrocystic leukodystrophy with micropolygyria. Structural modeling enabled us to make predictions about the effects of the mutations at the molecular level. Yeast and mammalian cell systems proved the pathogenic role of the mutant alleles by functional complementation in vivo. Nuclear-gene abnormalities causing mitochondrial translation defects represent a new, potentially broad field of mitochondrial medicine. Investigation of these defects is important to expand the molecular characterization of mitochondrial disorders and also may contribute to the elucidation of the complex control mechanisms, which regulate this fundamental pathway of mtDNA homeostasis.

Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy

Nemaline myopathy (NM) is a genetic muscle disorder characterized by muscle dysfunction and electron-dense protein accumulations (nemaline bodies) in myofibers. Pathogenic mutations have been described in 9 genes to date, but the genetic basis remains unknown in many cases. Here, using an approach that combined whole-exome sequencing (WES) and Sanger sequencing, we identified homozygous or compound heterozygous variants in LMOD3 in 21 patients from 14 families with severe, usually lethal, NM. LMOD3 encodes leiomodin-3 (LMOD3), a 65-kDa protein expressed in skeletal and cardiac muscle. LMOD3 was expressed from early stages of muscle differentiation; localized to actin thin filaments, with enrichment near the pointed ends; and had strong actin filament-nucleating activity. Loss of LMOD3 in patient muscle resulted in shortening and disorganization of thin filaments. Knockdown of lmod3 in zebrafish replicated NM-associated functional and pathological phenotypes. Together, these findings indicate that mutations in the gene encoding LMOD3 underlie congenital myopathy and demonstrate that LMOD3 is essential for the organization of sarcomeric thin filaments in skeletal muscle.

Muscle histone deacetylase 4 upregulation in amyotrophic lateral sclerosis: potential role in reinnervation ability and disease progression

Amyotrophic lateral sclerosis is a typically rapidly progressive neurodegenerative disorder affecting motor neurons leading to progressive muscle paralysis and death, usually from respiratory failure, in 3-5 years. Some patients have slow disease progression and prolonged survival, but the underlying mechanisms remain poorly understood. Riluzole, the only approved treatment, only modestly prolongs survival and has no effect on muscle function. In the early phase of the disease, motor neuron loss is initially compensated for by collateral reinnervation, but over time this compensation fails, leading to progressive muscle wasting. The crucial role of muscle histone deacetylase 4 and its regulator microRNA-206 in compensatory reinnervation and disease progression was recently suggested in a mouse model of amyotrophic lateral sclerosis (transgenic mice carrying human mutations in the superoxide dismutase gene). Here, we sought to investigate whether the microRNA-206-histone deacetylase 4 pathway plays a role in muscle compensatory reinnervation in patients with amyotrophic lateral sclerosis and thus contributes to disease outcome differences. We studied muscle reinnervation using high-resolution confocal imaging of neuromuscular junctions in muscle samples obtained from 11 patients with amyotrophic lateral sclerosis, including five long-term survivors. We showed that the proportion of reinnervated neuromuscular junctions was significantly higher in long-term survivors than in patients with rapidly progressive disease. We analysed the expression of muscle candidate genes involved in the reinnervation process and showed that histone deacetylase 4 upregulation was significantly greater in patients with rapidly progressive disease and was negatively correlated with the extent of muscle reinnervation and functional outcome. Conversely, the proposed regulator of histone deacetylase 4, microRNA-206, was upregulated in both patient groups, but did not correlate with disease progression or reinnervation. We conclude that muscle expression of histone deacetylase 4 may be a key factor for muscle reinnervation and disease progression in patients with amyotrophic lateral sclerosis. Specific histone deacetylase 4 inhibitors may then constitute a therapeutic approach to enhancing motor performance and slowing disease progression in amyotrophic lateral sclerosis.

A new muscle glycogen storage disease associated with glycogenin‐1 deficiency

We describe a slowly progressive myopathy in 7 unrelated adult patients with storage of polyglucosan in muscle fibers. Genetic investigation revealed homozygous or compound heterozygous deleterious variants in the glycogenin‐1 gene (GYG1). Most patients showed depletion of glycogenin‐1 in skeletal muscle, whereas 1 showed presence of glycogenin‐1 lacking the C‐terminal that normally binds glycogen synthase. Our results indicate that either depletion of glycogenin‐1 or impaired interaction with glycogen synthase underlies this new form of glycogen storage disease that differs from a previously reported patient with GYG1 mutations who showed profound glycogen depletion in skeletal muscle and accumulation of glycogenin‐1. Ann Neurol 2014;76:891–898

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.

An integrated diagnosis strategy for congenital myopathies.

Congenital myopathies are severe muscle disorders affecting adults as well as children in all populations. The diagnosis of congenital myopathies is constrained by strong clinical and genetic heterogeneity. Moreover, the majority of patients present with unspecific histological features, precluding purposive molecular diagnosis and demonstrating the need for an alternative and more efficient diagnostic approach. We used exome sequencing complemented by histological and ultrastructural analysis of muscle biopsies to identify the causative mutations in eight patients with clinically different skeletal muscle pathologies, ranging from a fatal neonatal myopathy to a mild and slowly progressive myopathy with adult onset. We identified RYR1 (ryanodine receptor) mutations in six patients and NEB (nebulin) mutations in two patients. We found novel missense and nonsense mutations, unraveled small insertions/deletions and confirmed their impact on splicing and mRNA/protein stability. Histological and ultrastructural findings of the muscle biopsies of the patients validated the exome sequencing results. We provide the evidence that an integrated strategy combining exome sequencing with clinical and histopathological investigations overcomes the limitations of the individual approaches to allow a fast and efficient diagnosis, accelerating the patient’s access to a better healthcare and disease management. This is of particular interest for the diagnosis of congenital myopathies, which involve very large genes like RYR1 and NEB as well as genetic and phenotypic heterogeneity.

Muscle histopathology in nebulin-related nemaline myopathy: ultrastrastructural findings correlated to disease severity and genotype.

Nemaline myopathy (NM) is a rare congenital myopathy characterised by hypotonia, muscle weakness, and often skeletal muscle deformities with the presence of nemaline bodies (rods) in the muscle biopsy. The nebulin (NEB) gene is the most commonly mutated and is thought to account for approximately 50% of genetically diagnosed cases of NM. We undertook a detailed muscle morphological analysis of 14 NEB-mutated NM patients with different clinical forms to define muscle pathological patterns and correlate them with clinical course and genotype. Three groups were identified according to clinical severity. Group 1 (n = 5) comprises severe/lethal NM and biopsy in the first days of life. Group 2 (n = 4) includes intermediate NM and biopsy in infancy. Group 3 (n = 5) comprises typical/mild NM and biopsy in childhood or early adult life. Biopsies underwent histoenzymological, immunohistochemical and ultrastructural analysis. Fibre type distribution patterns, rod characteristics, distribution and localization were investigated. Contractile performance was studied in muscle fibre preparations isolated from seven muscle biopsies from each of the three groups. G1 showed significant myofibrillar dissociation and smallness with scattered globular rods in one third of fibres; there was no type 1 predominance. G2 presented milder sarcomeric dissociation, dispersed or clustered nemaline bodies, and type 1 predominance/uniformity. In contrast, G3 had well-delimited clusters of subsarcolemmal elongated rods and type 1 uniformity without sarcomeric alterations. In accordance with the clinical and morphological data, functional studies revealed markedly low forces in muscle bundles from G1 and a better contractile performance in muscle bundles from biopsies of patients from G2, and G3.In conclusion NEB-mutated NM patients present a wide spectrum of morphological features. It is difficult to establish firm genotype phenotype correlation. Interestingly, there was a correlation between clinical severity on the one hand and the degree of sarcomeric dissociation and contractility efficiency on the other. By contrast the percentage of fibres occupied by rods, as well as the quantity and the sub sarcolemmal position of rods, appears to inversely correlate with severity. Based on our observations, we propose myofibrillar dissociation and changes in contractility as an important cause of muscle weakness in NEB-mutated NM patients.

High risk of severe cardiac adverse events in patients with mitochondrial m.3243A>G mutation.

ABSTRACT Objectives: To determine the long-term incidence of cardiac life-threatening complications and death in patients with the m.3243A>G mutation, and to identify cardiac prognostic factors. Methods: We retrospectively included patients carrying the m.3243A>G mutation who were admitted to the Neuromuscular Disease Clinic of Pitié Salpêtrière Hospital between January 1992 and December 2010. We collected information relative to their yearly neurologic and cardiac investigations, their mutation load in blood, urine, and muscle at initial admission, and the occurrence of cardiac life-threatening adverse events and death during follow-up. Results: Forty-one patients (median age = 47 years [36–55 years], men = 13) were included, of whom 38 had clinical manifestations of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) and 3 were asymptomatic. One patient had a personal history of cardiac transplantation. Cardiac investigations displayed left ventricular hypertrophy, left ventricular dysfunction, or both abnormalities in 18 patients, along with Wolff-Parkinson-White syndrome in 7, conduction system disease in 4, and atrial fibrillation in 1. Over a median 5-year (3–9 years) follow-up period, 11 patients died, including 3 due to heart failure; 7 had life-threatening adverse events, including 6 hospitalizations for severe heart failure and 1 resuscitated cardiac arrest. By multivariate analysis, left ventricular hypertrophy was the only parameter independently associated with occurrence of cardiac adverse events. Conclusion: Patients with the m.3243A>G mutation have a high incidence of cardiac death and life-threatening adverse events. Left ventricular hypertrophy was the only parameter independently associated with occurrence of these events.

A Premature Stop Codon in MYO18B is Associated with Severe Nemaline Myopathy with Cardiomyopathy

Abstract Background: Nemaline myopathies (NM) are rare and severe muscle diseases characterized by the presence of nemaline bodies (rods) in muscle fibers. Although ten genes have been implicated in the etiology of NM, an important number of patients remain without a molecular diagnosis. Objective: Here we describe the clinical and histopathological features of a sporadic case presenting with severe NM and cardiomyopathy. Using exome sequencing, we aimed to identify the causative gene. Results: We identified a homozygous nonsense mutation in the last exon of MYO18B, leading to a truncated protein lacking the most C-terminal part. MYO18B codes for an unconventional myosin protein and it is mainly expressed in skeletal and cardiac muscles, two tissues severely affected in the patient. We showed that the mutation does not impact on mRNA stability. Immunostaining and Western blot confirmed the absence of the full-length protein. Conclusion: We propose MYO18B as a novel gene associated with nemaline myopathy and cardiomyopathy.

Skeletal muscle biopsy analysis in reducing body myopathy and other FHL1-related disorders.

FHL1 mutations have been associated with various disorders that include reducing body myopathy (RBM), Emery-Dreifuss-like muscular dystrophy, isolated hypertrophic cardiomyopathy, and some overlapping conditions. We report a detailed histochemical, immunohistochemical, electron microscopic, and immunoelectron microscopic analyses of muscle biopsies from 18 patients carrying mutations in FHL1: 14 RBM patients (Group 1), 3 Emery-Dreifuss muscular dystrophy patients (Group 2), and 1 patient with hypertrophic cardiomyopathy and muscular hypertrophy (Group 2). Group 1 muscle biopsies consistently showed RBs associated with cytoplasmic bodies. The RBs showed prominent FHL1 immunoreactivity whereas desmin, αB-crystallin, and myotilin immunoreactivity surrounded RBs. By electron microscopy, RBs were composed of electron-dense tubulofilamentous material that seemed to spread progressively between the myofibrils and around myonuclei. By immunoelectron microscopy, FHL1 protein was found exclusively inside RBs. Group 2 biopsies showed mild dystrophic abnormalities without RBs; only minor nonspecific myofibrillar abnormalities were observed under electron microscopy. Molecular analysis revealed missense mutations in the second FHL1 LIM domain in Group 1 patients and ins/del or missense mutations within the fourth FHL1 LIM domain in Group 2 patients. Our findings expand the morphologic features of RBM, clearly demonstrate the localization of FHL1 in RBs, and further illustrate major morphologic differences among different FHL1-related myopathies.