Prominent muscle involvement in a familial form of mitochondrial disease due to a COA8 variant.

Isolated mitochondrial respiratory chain Complex IV (Cytochrome c Oxidase or COX) deficiency is the second most frequent isolated respiratory chain defect. Causative mutations are mainly identified in structural COX subunits or in proteins involved in the maturation and assembly of the COX holocomplex. We describe an Italian familial case of mitochondrial myopathy due to a variant in the COX assembly factor 8 gene (COA8). Patient 1 is a 52-year-old woman who presented generalized epilepsy and retinitis pigmentosa at 10 years of age. From her early adulthood she complained about cramps and myalgia after exercise, and bilateral hearing loss emerged. Last neurological examination (52 years of age) showed bilateral ptosis, muscle weakness, peripheral neuropathy, mild dysarthria and dysphonia, cognitive impairment. Muscle biopsy had shown the presence of ragged-red fibers. Patient 2 (Patient 1's sister) is a 53-year-old woman presenting fatigability, myalgia, and hearing loss. Neurological examination showed ptosis and muscle weakness. Muscle biopsy displayed a diffuse reduction of COX activity staining and ragged-red fibers. Both sisters presented secondary amenorrhea. After ruling out mtDNA mutations, Whole Exome Sequencing analysis identified the novel homozygous COA8 defect c.170_173dupGACC, p.(Pro59fs) in the probands. Loss-of-function COA8 mutations have been associated with cavitating leukoencephalopathy with COX deficiency in 9 reported individuals. Disease course shows an early-onset rapid clinical deterioration, affecting both cognitive and motor functions over months, followed by stabilization and slow improvement over several years. Our findings expand the clinical spectrum of COA8-related disease. We confirm the benign course of this rare disorder, highlighting its (intrafamilial) clinical variability.

Lafora Disease: A Case Report and Evolving Treatment Advancements.

Lafora disease is a rare genetic disorder characterized by a disruption in glycogen metabolism. It manifests as progressive myoclonus epilepsy and cognitive decline during adolescence. Pathognomonic is the presence of abnormal glycogen aggregates that, over time, produce large inclusions (Lafora bodies) in various tissues. This study aims to describe the clinical and histopathological aspects of a novel Lafora disease patient, and to provide an update on the therapeutical advancements for this disorder. A 20-year-old Libyan boy presented with generalized tonic-clonic seizures, sporadic muscular jerks, eyelid spasms, and mental impairment. Electroencephalography showed multiple discharges across both brain hemispheres. Brain magnetic resonance imaging was unremarkable. Muscle biopsy showed increased lipid content and a very mild increase of intermyofibrillar glycogen, without the polyglucosan accumulation typically observed in Lafora bodies. Despite undergoing three lines of antiepileptic treatment, the patient's condition showed minimal to no improvement. We identified the homozygous variant c.137G>A, p.(Cys46Tyr), in the EPM2B/NHLRC1 gene, confirming the diagnosis of Lafora disease. To our knowledge, the presence of lipid aggregates without Lafora bodies is atypical. Lafora disease should be considered during the differential diagnosis of progressive, myoclonic, and refractory epilepsies in both children and young adults, especially when accompanied by cognitive decline. Although there are no effective therapies yet, the development of promising new strategies prompts the need for an early and accurate diagnosis.

The Profiling of 179 miRNA Expression in Serum from Limb Girdle Muscular Dystrophy Patients and Healthy Controls.

Limb girdle muscular dystrophies (LGMDs) are a group of genetically inherited neuromuscular diseases with a very variable clinical presentation and overlapping traits. Over the last few years there has been an increasing interest in the use of non-invasive circulating biomarkers to monitor disease progression and to evaluate the efficacy of therapeutic approaches. Our aim was to identify the miRNA signature with potential value for LGMD patient screening and stratification. Using miRCURY LNA miRNA qPCR Serum/Plasma Panel, we analyzed 179 miRNAs from 16 patients, divided in four pools based on their genetic diagnosis, and from healthy controls. The miRNAs analysis showed a total of 107 dysregulated miRNAs in LGMD patients when compared to the healthy controls. After filtering via skeletal tissue expression and gene/pathways target analysis, the number of dysregulated miRNAs drastically reduced. Six selected miRNAs-let-7f-5p (in LGMDR1), miR-20a-5p (in LGMDR2), miR-130b-5p, miR-378a-5p (both in LGMDR3), miR-376c-3p and miR-382-5p (both in LGMDR4)-whose expression was significantly lower compared to controls in the different LGMD pools, were further investigated. The bioinformatic analysis of the target genes in each selected miRNA revealed ECM-receptor interaction and TGF-beta signaling as the most involved pathways. The correlation analysis showed a good correlation of let-7f-5p with fibrosis and with the cross sectional area of type I and type II fibers, while miR-130b-5p showed a good correlation with the age of onset of the disease. The receiver operating characteristic curves showed how single miRNAs were able to discriminate a specific group of LGMD patients and how the combination of six miRNAs was able to discriminate LGMD patients from controls.

Characterization of Skeletal Muscle Biopsy and Derived Myoblasts in a Patient Carrying Arg14del Mutation in Phospholamban Gene.

Phospholamban is involved in the regulation of the activity and storage of calcium in cardiac muscle. Several mutations have been identified in the PLN gene causing cardiac disease associated with arrhythmogenic and dilated cardiomyopathy. The patho-mechanism underlying PLN mutations is not fully understood and a specific therapy is not yet available. PLN mutated patients have been deeply investigated in cardiac muscle, but very little is known about the effect of PLN mutations in skeletal muscle. In this study, we investigated both histological and functional features in skeletal muscle tissue and muscle-derived myoblasts from an Italian patient carrying the Arg14del mutation in PLN. The patient has a cardiac phenotype, but he also reported lower limb fatigability, cramps and fasciculations. The evaluation of a skeletal muscle biopsy showed histological, immunohistochemical and ultrastructural alterations. In particular, we detected an increase in the number of centronucleated fibers and a reduction in the fiber cross sectional area, an alteration in p62, LC3 and VCP proteins and the formation of perinuclear aggresomes. Furthermore, the patient's myoblasts showed a greater propensity to form aggresomes, even more marked after proteasome inhibition compared with control cells. Further genetic and functional studies are necessary to understand whether a definition of PLN myopathy, or cardiomyopathy plus, can be introduced for selected cases with clinical evidence of skeletal muscle involvement. Including skeletal muscle examination in the diagnostic process of PLN-mutated patients can help clarify this issue.

Distribution of Exonic Variants in Glycogen Synthesis and Catabolism Genes in Late Onset Pompe Disease (LOPD).

Pompe disease (PD) is a monogenic autosomal recessive disorder caused by biallelic pathogenic variants of the GAA gene encoding lysosomal alpha-glucosidase; its loss causes glycogen storage in lysosomes, mainly in the muscular tissue. The genotype-phenotype correlation has been extensively discussed, and caution is recommended when interpreting the clinical significance of any mutation in a single patient. As there is no evidence that environmental factors can modulate the phenotype, the observed clinical variability in PD suggests that genetic variants other than pathogenic GAA mutations influence the mechanisms of muscle damage/repair and the overall clinical picture. Genes encoding proteins involved in glycogen synthesis and catabolism may represent excellent candidates as phenotypic modifiers of PD. The genes analyzed for glycogen synthesis included UGP2, glycogenin (GYG1-muscle, GYG2, and other tissues), glycogen synthase (GYS1-muscle and GYS2-liver), GBE1, EPM2A, NHLRC1, GSK3A, and GSK3B. The only enzyme involved in glycogen catabolism in lysosomes is α-glucosidase, which is encoded by GAA, while two cytoplasmic enzymes, phosphorylase (PYGB-brain, PGL-liver, and PYGM-muscle) and glycogen debranching (AGL) are needed to obtain glucose 1-phosphate or free glucose. Here, we report the potentially relevant variants in genes related to glycogen synthesis and catabolism, identified by whole exome sequencing in a group of 30 patients with late-onset Pompe disease (LOPD). In our exploratory analysis, we observed a reduced number of variants in the genes expressed in muscles versus the genes expressed in other tissues, but we did not find a single variant that strongly affected the phenotype. From our work, it also appears that the current clinical scores used in LOPD do not describe muscle impairment with enough qualitative/quantitative details to correlate it with genes that, even with a slightly reduced function due to genetic variants, impact the phenotype.

Using Cluster Analysis to Overcome the Limits of Traditional Phenotype-Genotype Correlations: The Example of RYR1-Related Myopathies.

Thanks to advances in gene sequencing, RYR1-related myopathy (RYR1-RM) is now known to manifest itself in vastly heterogeneous forms, whose clinical interpretation is, therefore, highly challenging. We set out to develop a novel unsupervised cluster analysis method in a large patient population. The objective was to analyze the main RYR1-related characteristics to identify distinctive features of RYR1-RM and, thus, offer more precise genotype-phenotype correlations in a group of potentially life-threatening disorders. We studied 600 patients presenting with a suspicion of inherited myopathy, who were investigated using next-generation sequencing. Among them, 73 index cases harbored variants in RYR1. In an attempt to group genetic variants and fully exploit information derived from genetic, morphological, and clinical datasets, we performed unsupervised cluster analysis in 64 probands carrying monoallelic variants. Most of the 73 patients with positive molecular diagnoses were clinically asymptomatic or pauci-symptomatic. Multimodal integration of clinical and histological data, performed using a non-metric multi-dimensional scaling analysis with k-means clustering, grouped the 64 patients into 4 clusters with distinctive patterns of clinical and morphological findings. In addressing the need for more specific genotype-phenotype correlations, we found clustering to overcome the limits of the "single-dimension" paradigm traditionally used to describe genotype-phenotype relationships.

MERRF Mutation A8344G in a Four-Generation Family without Central Nervous System Involvement: Clinical and Molecular Characterization.

A 53-year-old man approached our Neuromuscular Unit following an incidental finding of hyperckemia. Similar to his mother who had died at the age of 77 years, he was diabetic and had a few lipomas. The patient's two sisters, aged 60 and 50 years, did not have any neurological symptoms. Proband's skeletal muscle biopsy showed several COX-negative fibers, many of which were "ragged red". Genetic analysis revealed the presence of the A8344G mtDNA mutation, which is most commonly associated with a maternally inherited multisystem mitochondrial disorder known as MERRF (myoclonus epilepsy with ragged-red fibers). The two sisters also carry the mutation. Family members on the maternal side were reported healthy. Although atypical phenotypes have been reported in association with the A8344G mutation, central nervous system (CSN) manifestations other than myoclonic epilepsy are always reported in the family tree. If present, our four-generation family manifestations are late-onset and do not affect CNS. This could be explained by the fact that the mutational load remains low and therefore prevents tissues/organs from reaching the pathologic threshold. The fact that this occurs throughout generations and that CNS, which has the highest energetic demand, is clinically spared, suggests that regulatory genes and/or pathways affect mitochondrial segregation and replication, and protect organs from progressive dysfunction.

NEB mutations disrupt the super-relaxed state of myosin and remodel the muscle metabolic proteome in nemaline myopathy.

Nemaline myopathy (NM) is one of the most common non-dystrophic genetic muscle disorders. NM is often associated with mutations in the NEB gene. Even though the exact NEB-NM pathophysiological mechanisms remain unclear, histological analyses of patients' muscle biopsies often reveal unexplained accumulation of glycogen and abnormally shaped mitochondria. Hence, the aim of the present study was to define the exact molecular and cellular cascade of events that would lead to potential changes in muscle energetics in NEB-NM. For that, we applied a wide range of biophysical and cell biology assays on skeletal muscle fibres from NM patients as well as untargeted proteomics analyses on isolated myofibres from a muscle-specific nebulin-deficient mouse model. Unexpectedly, we found that the myosin stabilizing conformational state, known as super-relaxed state, was significantly impaired, inducing an increase in the energy (ATP) consumption of resting muscle fibres from NEB-NM patients when compared with controls or with other forms of genetic/rare, acquired NM. This destabilization of the myosin super-relaxed state had dynamic consequences as we observed a remodeling of the metabolic proteome in muscle fibres from nebulin-deficient mice. Altogether, our findings explain some of the hitherto obscure hallmarks of NM, including the appearance of abnormal energy proteins and suggest potential beneficial effects of drugs targeting myosin activity/conformations for NEB-NM.

A Schematic Approach to Defining the Prevalence of COL VI Variants in Five Years of Next-Generation Sequencing.

OBJECTIVE: To define the prevalence of variants in collagen VI genes through a next-generation sequencing (NGS) approach in undiagnosed patients with suspected neuromuscular disease and to propose a diagnostic flowchart to assess the real pathogenicity of those variants. METHODS: In the past five years, we have collected clinical and molecular information on 512 patients with neuromuscular symptoms referred to our center. To pinpoint variants in COLVI genes and corroborate their real pathogenicity, we sketched a multistep flowchart, taking into consideration the bioinformatic weight of the gene variants, their correlation with clinical manifestations and possible effects on protein stability and expression. RESULTS: In Step I, we identified variants in COLVI-related genes in 48 patients, of which three were homozygous variants (Group 1). Then, we sorted variants according to their CADD score, clinical data and complementary studies (such as muscle and skin biopsy, study of expression of COLVI on fibroblast or muscle and muscle magnetic resonance). We finally assessed how potentially pathogenic variants (two biallelic and 12 monoallelic) destabilize COL6A1-A2-A3 subunits. Overall, 15 out of 512 patients were prioritized according to this pipeline. In seven of them, we confirmed reduced or absent immunocytochemical expression of collagen VI in cultured skin fibroblasts or in muscle tissue. CONCLUSIONS: In a real-world diagnostic scenario applied to heterogeneous neuromuscular conditions, a multistep integration of clinical and molecular data allowed the identification of about 3% of those patients harboring pathogenetic collagen VI variants.

Expanding the phenotypic spectrum of non-alcoholic fatty liver disease and hypertriglyceridemia.

Background and aims: Hypertriglyceridemia is a common feature of metabolic syndrome (MetS), as well as of non-alcoholic fatty liver disease (NAFLD), which is considered the hepatic manifestation of MetS. Fat accumulation in hepatocytes may alter mitochondrial homeostasis predisposing to advanced liver disease. Here, we report a case of a 40-year-old woman with early aggressive NAFLD due to severe hypertriglyceridemia that ensued from a combination of genetic variants and additional metabolic risk factors. Methods: Genetic screening was performed by using whole-exome sequencing (WES), and mitochondrial structures were evaluated by TEM. Results: At presentation, the patient is reported to have hepatomegaly, hypertriglyceridemia, and raised transaminases. Genetic analysis revealed that the patient beard heritable alterations in genes implicated in lipid handling, among which APOB, APOE, CETP, and HSPG2, accompanied by missense mutations in genes involved in mitochondrial function, i.e., AK2, ALG6, ASPA, NDUFAF1, POLG, and TMEM70. Abdominal ultrasound (US) and transient elastography were suggestive of severe hepatic steatosis and fibrosis. A liver biopsy confirmed the diagnosis of non-alcoholic steatohepatitis (NASH)-related fibrosis. Thus, to better outline whether mutations involved in lipid remodeling and mitochondrial function may also affect organelles' morphology, we exploited TEM. Along with multifaceted abnormalities of mitochondrial architecture that have been already observed in patients with NAFLD, astonishing ultrastructural defects, such as mitochondrial vacuolization, sub-compartmentalization, and onion-like mitochondria, were identified. Conclusion: The anomalies reported may expand the phenotypic spectrum of mitochondrial abnormalities observed in patients with NAFLD, which may contribute to the switching toward a progressive disease.

Immunofluorescence signal intensity measurements as a semi-quantitative tool to assess sarcoglycan complex expression in muscle biopsy.

Sarcoglycanopathies are highly heterogeneous in terms of disease progression, muscular weakness, loss of ambulation and cardiac/respiratory involvement. Their clinical severity usually correlates with the residual protein amount, which makes protein quantification extremely relevant. Sarcoglycanopathy diagnosis is genetic, but skeletal muscle analysis - by both immunohistochemistry and Western blot (WB) - is still mandatory to establish the correct diagnostic process. Unfortunately, however, WB analysis cannot be performed if the bioptic specimen is scarce. This study provides a sensitive tool for semi-quantification of residual amount of sarcoglycans in patients affected by sarcoglycanopathies, based on immunofluorescence staining on skeletal muscle sections, image acquisition and software elaboration. We applied this method to eleven sarcoglycanopathies, seven Becker muscular dystrophies and four age-matched controls. Fluorescence data analysed in patients and compared to age-matched controls showed a significant reduction of the mutated sarcoglycan expression and a variable reduction of the other sarcoglycans. Fluorescence normalized data analysed in relation to the age of onset of the disease, showed a negative correlation of α-sarcoglycan fluorescent signal versus fibrosis in patients with an early age of onset and a negative correlation between δ-sarcoglycan signal and fibrosis in both intermediate and late age of onset groups. The availability of a method that allows objective quantification of the sarcolemmal proteins, faster and less consuming than WB analysis and able to detect low residual sarcoglycan expression with great sensitivity, proves useful to better define both patient prognosis and expected disease evolution. The proposed method could be employed also to monitor the efficacy of therapeutic interventions and during clinical trials.

Antisense Morpholino-Based In Vitro Correction of a Pseudoexon-Generating Variant in the SGCB Gene.

Limb-girdle muscular dystrophies (LGMD) are clinically and genetically heterogenous presentations displaying predominantly proximal muscle weakness due to the loss of skeletal muscle fibers. Beta-sarcoglycanopathy (LGMDR4) results from biallelic molecular defects in SGCB and features pediatric onset with limb-girdle involvement, often complicated by respiratory and heart dysfunction. Here we describe a patient who presented at the age of 12 years reporting high creatine kinase levels and onset of cramps after strenuous exercise. Instrumental investigations, including a muscle biopsy, pointed towards a diagnosis of beta-sarcoglycanopathy. NGS panel sequencing identified two variants in the SGCB gene, one of which (c.243+1548T>C) was found to promote the inclusion of a pseudoexon between exons 2 and 3 in the SGCB transcript. Interestingly, we detected the same genotype in a previously reported LGMDR4 patient, deceased more than twenty years ago, who had escaped molecular diagnosis so far. After the delivery of morpholino oligomers targeting the pseudoexon in patient-specific induced pluripotent stem cells, we observed the correction of the physiological splicing and partial restoration of protein levels. Our findings prompt the analysis of the c.243+1548T>C variant in suspected LGMDR4 patients, especially those harbouring monoallelic SGCB variants, and provide a further example of the efficacy of antisense technology for the correction of molecular defects resulting in splicing abnormalities.

Case Reports: Novel Missense Variants in the Filamin C Actin Binding Domain Cause Variable Phenotypes.

Filamin C is a large dimeric actin-binding protein, most prevalent in skeletal and cardiac muscle Z-discs, where it participates in sarcomere mechanical stabilization and intracellular signaling, interacting with numerous binding partners. Dominant heterozygous mutations of Filamin C gene cause several forms of myopathy and structural or arrhythmogenic cardiomyopathy. In this report we describe clinical and molecular findings of two Italian patients, in whom we identified two novel missense variants located within the Filamin C actin binding domain. Muscle imaging, histological and ultrastructural findings are also reported. Our results underline the extreme inter- and intrafamilial variability of clinical manifestations, hence the need to extend the investigation also to asymptomatic relatives, and the relevance of a broad diagnostic approach involving muscle electron microscopy, skeletal muscle magnetic resonance imaging and next generation sequencing techniques.

Expanding the Phenotypic Spectrum of Vocal Cord and Pharyngeal Weakness With Distal Myopathy due to the p.S85C MATR3 Mutation.

Objectives: The c.254C>G (p.S85C) MATR3 variant causes vocal cord and pharyngeal weakness with distal myopathy (VCPDM), which is characterized by progressive, asymmetric, predominantly distal muscle weakness, dysphonia, dysphagia, and respiratory impairment. Herein, we describe an Italian patient who harbored the p.S85C MATR3 variant and showed a composite phenotype of VCPDM and sensorimotor polyneuropathy. Methods: The proband underwent neurologic evaluation, muscular MRI of the lower limbs, neurophysiologic assessment, muscle biopsy, and spirometry. After excluding common acquired and genetic causes of sensorimotor polyneuropathy, a larger group of genes involved in inherited forms of neuropathy, distal myopathy, and motor neuron disorders were analyzed by next-generation sequencing targeted panels. Results: The patient, affected by progressive distal muscle weakness and hypotrophy, myalgias, dysphonia, dysphagia, respiratory impairment, and sensory abnormalities, harbored the heterozygous c.254C>G (p.S85C) MATR3 substitution. Neurophysiologic assessment revealed a severe sensorimotor polyneuropathy. Variation of fiber size, central nuclei, and nonrimmed vacuoles were evident at muscle biopsy. Discussion: This finding extends the MATR3-associated VCPDM phenotypic spectrum and suggests considering MATR3 analysis in suspected congenital polyneuropathies with odd features, including dysphonia, dysphagia, and respiratory insufficiency.

A new phenotype of muscle glycogen synthase deficiency (GSD0B) characterized by an adult onset myopathy without cardiomyopathy.

Muscle Glycogenosis type 0 (GSD0B) is an extremely rare disorder first recognized in 2007 in three siblings with childhood onset and severe cardiomyopathy. Since then, a few cases with severe cardiac involvement and premature death have been reported. We describe two unrelated cases presenting with an adult-onset myopathy with no heart involvement. Clinical features were quite similar in both patients, mainly characterized by early fatigability, myalgia and muscle weakness. Muscle biopsy revealed marked glycogen depletion in nearly all myofibers. Biochemical assay demonstrated a marked reduction of Glycogen Synthase (GS) activity. Sequence analysis of GYS1 revealed two new variants: a homozygous G to C substitution in the splice donor consensus site (c.678+1G>C) in patient1 and a homozygous missense variant c.630G>C in exon 3 (p. Asp145His) in patient 2. This study describes a new phenotype of muscle GSD0B presenting with adult onset, proximal myopathy, no cardiac abnormalities and a quite benign disease course. This report highlights the importance of a systematic diagnostic approach that includes muscle morphology and enzymatic assay to facilitate the identification of adult patients with GSD0B.

Muscle histological changes in a large cohort of patients affected with Becker muscular dystrophy.

Becker muscular dystrophy (BMD) is a severe X-linked muscle disease. Age of onset, clinical variability, speed of progression and affected tissues display wide variability, making a clinical trial design for drug development very complex. The histopathological changes in skeletal muscle tissue are central to the pathogenesis, but they have not been thoroughly elucidated yet. Here we analysed muscle biopsies from a large cohort of BMD patients, focusing our attention on the histopathological muscle parameters, as fibrosis, fatty replacement, fibre cross sectional area, necrosis, regenerating fibres, splitting fibres, internalized nuclei and dystrophy evaluation. We correlated histological parameters with both demographic features and clinical functional evaluations. The most interesting results of our study are the accurate quantification of fibroadipose tissue replacement and the identification of some histopathological aspects that well correlate with clinical performances. Through correlation analysis, we divided our patients into three clusters with well-defined histological and clinical features. In conclusion, this is the first study that analyses in detail the histological characteristics of muscle biopsies in a large cohort of BMD patients, correlating them to a functional impairment. The collection of these data help to better understand the histopathological progression of the disease and can be useful to validate any pharmacological trial in which the modification of muscle biopsy is utilized as outcome measure.

Cell environment shapes TDP-43 function with implications in neuronal and muscle disease.

TDP-43 (TAR DNA-binding protein 43) aggregation and redistribution are recognised as a hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. As TDP-43 inclusions have recently been described in the muscle of inclusion body myositis patients, this highlights the need to understand the role of TDP-43 beyond the central nervous system. Using RNA-seq, we directly compare TDP-43-mediated RNA processing in muscle (C2C12) and neuronal (NSC34) mouse cells. TDP-43 displays a cell-type-characteristic behaviour targeting unique transcripts in each cell-type, which is due to characteristic expression of RNA-binding proteins, that influence TDP-43's performance and define cell-type specific splicing. Among splicing events commonly dysregulated in both cell lines, we identify some that are TDP-43-dependent also in human cells. Inclusion levels of these alternative exons are altered in tissues of patients suffering from FTLD and IBM. We therefore propose that TDP-43 dysfunction contributes to disease development either in a common or a tissue-specific manner.

Clinical and genetic features of a cohort of patients with MFN2-related neuropathy.

Charcot-Marie-Tooth disease type 2A (CMT2A) is a rare inherited axonal neuropathy caused by mutations in MFN2 gene, which encodes Mitofusin 2, a transmembrane protein of the outer mitochondrial membrane. We performed a cross-sectional analysis on thirteen patients carrying mutations in MFN2, from ten families, describing their clinical and genetic characteristics. Evaluated patients presented a variable age of onset and a wide phenotypic spectrum, with most patients presenting a severe phenotype. A novel heterozygous missense variant was detected, p.K357E. It is located at a highly conserved position and predicted as pathogenic by in silico tools. At a clinical level, the p.K357E carrier shows a severe sensorimotor axonal neuropathy. In conclusion, our work expands the genetic spectrum of CMT2A, disclosing a novel mutation and its related clinical effect, and provides a detailed description of the clinical features of a cohort of patients with MFN2 mutations. Obtaining a precise genetic diagnosis in affected families is crucial both for family planning and prenatal diagnosis, and in a therapeutic perspective, as we are entering the era of personalized therapy for genetic diseases.

Genetic defects are common in myopathies with tubular aggregates.

OBJECTIVE: A group of genes have been reported to be associated with myopathies with tubular aggregates (TAs). Many cases with TAs still lack of genetic clarification. This study aims to explore the genetic background of cases with TAs in order to improve our knowledge of the pathogenesis of these rare pathological structures. METHODS: Thirty-three patients including two family members with biopsy confirmed TAs were collected. Whole-exome sequencing was performed on 31 unrelated index patients and a candidate gene search strategy was conducted. The identified variants were confirmed by Sanger sequencing. The wild-type and the mutant p.Ala11Thr of ALG14 were transfected into human embryonic kidney 293 cells (HEK293), and western blot analysis was performed to quantify protein expression levels. RESULTS: Eleven index cases (33%) were found to have pathogenic variant or likely pathogenic variants in STIM1, ORAI1, PGAM2, SCN4A, CASQ1 and ALG14. Among them, the c.764A>T (p.Glu255Val) in STIM1 and the c.1333G>C (p.Val445Leu) in SCN4A were novel. Western blot analysis showed that the expression of ALG14 protein was severely reduced in the mutant ALG14 HEK293 cells (p.Ala11Thr) compared with wild type. The ALG14 variants might be associated with TAs in patients with complex multisystem disorders. INTERPRETATION: This study expands the phenotypic and genotypic spectrums of myopathies with TAs. Our findings further confirm previous hypothesis that genes related with calcium signalling pathway and N-linked glycosylation pathway are the main genetic causes of myopathies with TAs.

Polymorphism in exercise genes and respiratory function in late-onset Pompe disease.

Genetic polymorphisms influencing muscle structure and metabolism may affect the phenotype of metabolic myopathies. We here analyze the possible influence of a wide panel of "exercise genes" on the severity and progression of respiratory dysfunction in late-onset Pompe disease (LOPD). We stratified patients with comparable age and disease duration according to the severity of their respiratory phenotype, assessed by both upright FVC% and postural drop in FVC%. We included 43 patients with LOPD (25 males, age 50.8 ± 13.6 yr) with a 2-yr follow-up since the beginning of enzyme replacement therapy (ERT). Twenty-two patients showed a postural drop >25% T0, seven other patients developed it during the follow-up. We analyzed the relationship between the progression of respiratory dysfunction and genetic polymorphisms affecting muscle function and structure [angiotensin converting enzyme (ACE), α-actinin 3 (ACTN3), peroxisome proliferator-activated receptor α (PPR-α), angiotensin (AGT)], glycogen metabolism [glycogen synthase (GYS), glycogen synthase kinase-3 isoform ß (GSK3ß)], and autophagy [sirtuin 1 (SIRT1), autophagy-related gene 7 (ATG7)]. Individuals carrying two copies of the ACE D-allele shared a 24-fold increase in the risk of severe respiratory dysfunction and progression during the 2-yr follow-up. ACTN3-XX polymorphism was also associated with worse respiratory outcome. The study of exercise genes is of particular interest in respiratory muscles, due to their peculiar features, that is, continuous, low-intensity contraction and prominent recruitment of type I fibers. In line with previous observations on skeletal muscles, ACE-DD and ACTN3-XX genotypes were associated with indirect evidence of more severe respiratory phenotypes. On the contrary, polymorphisms related to autophagy and glycogen metabolism did not seem to influence respiratory muscles.NEW & NOTEWORTHY Previous reports evaluated the role of exercise genes in influencing skeletal muscle phenotype and response to ERT in LOPD. Here, we investigate the role of polymorphisms in several exercise gene, focusing on respiratory muscles. ACE-DD and ACTN3-XX polymorphisms, possibly influencing muscle properties and fiber composition, were associated with more severe respiratory phenotypes.