MIOTONIA CONGENITA:CARATTERIZZAZIONE IN MODELLI IN VITRO DI MUTANTI DEL CANALE DEL CLORO MUSCOLARE CLC-1

Myotonia congenita (MC) belongs to the group of non-dystrophic myotonia and can be inherited either by an autosomal dominant (Thomsen\u2019s disease) or recessive manner (Becker\u2019s disease). It is characterized by impaired muscle relaxation after voluntary contraction and variable degrees of muscle weakness. MC is caused by mutations in the CLCN1 gene on chromosome 7q35 encoding the major skeletal muscle chloride channel CLC-1. It is well established that chloride channels play a role in the regulation of the muscle membrane and thus participate in the maintenance of the resting potential. Their inactivation by mutations modifies the cycle of excitability of the muscle membrane, shifting it towards hyperexcitability by slowing the return of the membrane to the resting potential after depolarization. Each muscle chloride channel comprises two identical protein molecules, each constituting a separate ion conductance pathway, the so-called protopore. In autosomal recessive myotonia congenita, both subunits have a disease-causing mutation. This results in chloride channel reduction to 40% or less, which is sufficient to cause myotonic contractions. Autosomal dominant myotonia congenita is believed to result from the presence of one dominant-negative mutation that modifies either the gating of both protopores or the selectivity of one of the two protopores. However, some mutations have been found to lead to autosomal dominant myotonia congenita in some patients, and to a homozygous recessive form in others. On clinical grounds, the dominant and recessive forms may be indistinguishable and the electromyography analysis does not distinguish between the recessive and the dominant phenotype. This study also confirmed the genetic heterogeneity of this condition, and suggested that the greater the number of pathogenic mutations described the more accurate will be the genetic counseling. We described 12 novel mutations: c.1606G>C (p.Val536Leu), c.2533G>A (p.Gly845Ser), c.2434C>T (p.Gln812X), c.1499G>T (p.Glu500X), c.1012C>T (p.Arg338X), c.2403+1G>A, c.2840T>A (p.Val947Glu), c.1598C>T (p.Thr533Ile), c.1110delC, c.590T>A (p.Ile197Arg), c.2276insA Fs800X, c.490T>C (p.Trp164Arg) in 22 unrelated Italian patients fitting the criteria for either Thomsen or Becker disease. To further understand the functional outcome of selected missense mutations found in nine patients (p.Trp164Arg, p.Ile197Arg, p.Gly845Ser, p.Val536Leu, p.Phe167Leu and the previously reported p.Gly190Ser) we characterized the biophysical properties of mutant ion channels in tsA cell model. In the physiological range of muscle membrane potential, all the tested mutations, excepting p.Gly845Ser, reduced the open probability, increased the fast and slow components of deactivation and affect pore properties. These defects in the physiology of hClC-1 channels were discussed in relation to the molecular background and the clinical features observed in myotonic patients. To verify the expression of these variants at RNA level, RealTime-PCR was performed on the studied missense mutations, and on the two nonsense p.Arg338X and p.Gln812X in tsA cell model. Results confirmed that none of these mutations, except p.Trp164Arg, changed significantly the expression of transcript amount compared to wild-type mRNAs. These data expand the spectrum of CLCN1 mutations and contribute to genotype-phenotype correlations. Furthermore, we provide insights into the protein structure of ClC-1 and its physiological role in the maintenance of membrane resting potential

Neuromuscular excitability changes produced by sustained voluntary contraction and response to mexiletine in myotonia congenita

Objective: To investigate the cause of transient weakness in myotonia congenita (MC) and the mechanism of action of mexiletine in reducing weakness. Methods: The changes in neuromuscular excitability produced by 1. min of maximal voluntary contractions (MVC) were measured on the amplitude of compound muscle action potentials (CMAP) in two patients with either recessive or dominant MC, compared to control values obtained in 20 healthy subjects. Measurements were performed again in MC patients after mexiletine therapy. Results: Transient reduction in maximal CMAP amplitude lasting several minutes after MVC was evident in MC patients, whereas no change was observed in controls. Mexiletine efficiently reduced this transient CMAP depression in both patients. Discussion: Transient CMAP depression following sustained MVC may represent the electrophysiological correlate of the weakness clinically experienced by the patients. In MC, the low chloride conductance could induce self-sustaining action potentials after MVC, determining progressive membrane depolarization and a loss of excitability of muscle fibers, thus resulting in transient paresis. Mexiletine may prevent conduction block due to excessive membrane depolarization, thus reducing the transient CMAP depression following sustained MVC

Spinal muscular atrophy phenotype is ameliorated in human motor neurons by SMN increase via different novel RNA therapeutic approaches

Spinal muscular atrophy (SMA) is a primary genetic cause of infant mortality due to mutations in the Survival Motor Neuron (SMN) 1 gene. No cure is available. Antisense oligonucleotides (ASOs) aimed at increasing SMN levels from the paralogous SMN2 gene represent a possible therapeutic strategy. Here, we tested in SMA human induced pluripotent stem cells (iPSCs) and iPSC-differentiated motor neurons, three different RNA approaches based on morpholino antisense targeting of the ISSN-1, exon-specific U1 small nuclear RNA (ExSpeU1), and Transcription Activator-Like Effector-Transcription Factor (TALE-TF). All strategies act modulating SMN2 RNA: ASO affects exon 7 splicing, TALE-TF increase SMN2 RNA acting on the promoter, while ExSpeU1 improves pre-mRNA processing. These approaches induced up-regulation of full-length SMN mRNA and differentially affected the Delta-7 isoform: ASO reduced this isoform, while ExSpeU1 and TALE-TF increased it. All approaches upregulate the SMN protein and significantly improve the in vitro SMA motor neurons survival. Thus, these findings demonstrate that therapeutic tools that act on SMN2 RNA are able to rescue the SMA disease phenotype. Our data confirm the feasibility of SMA iPSCs as in vitro disease models and we propose novel RNA approaches as potential therapeutic strategies for treating SMA and other genetic neurological disorders

The analysis of myotonia congenita mutations discloses functional clusters of amino acids within the CBS2 domain and the C-terminal peptide of the ClC-1 channel

Myotonia congenita (MC) is a skeletal-muscle hyperexcitability disorder caused by loss-of-function mutations in the ClC-1 chloride channel. Mutations are scattered over the entire sequence of the channel protein, with more than 30\ua0mutations located in the poorly characterized cytosolic C-terminal domain. In this study, we characterized, through patch clamp, seven ClC-1 mutations identified in patients affected by MC of various severities and located in the C-terminal region. The p.Val829Met, p.Thr832Ile, p.Val851Met, p.Gly859Val, and p.Leu861Pro mutations reside in the CBS2 domain, while p.Pro883Thr and p.Val947Glu are in the C-terminal peptide. We showed that the functional properties of mutant channels correlated with the clinical phenotypes of affected individuals. In addition, we defined clusters of ClC-1 mutations within CBS2 and C-terminal peptide subdomains that share the same functional defect: mutations between 829 and 835 residues and in residue 883 induced an alteration of voltage dependence, mutations between 851 and 859 residues, and in residue 947 induced a reduction of chloride currents, whereas mutations on 861 residue showed no obvious change in ClC-1 function. This study improves our understanding of the mechanisms underlying MC, sheds light on the role of the C-terminal region in ClC-1 function, and provides information to develop new antimyotonic drugs

Glucose-free/high-protein diet improves hepatomegaly and exercise intolerance in glycogen storage disease type III mice

Glycogen disease type III (GSDIII), a rare incurable autosomal recessive disorder due to glycogen debranching enzyme deficiency, presents with liver, heart and skeletal muscle impairment, hepatomegaly and ketotic hypoglycemia. Muscle weakness usually worsens to fixed myopathy and cardiac involvement may present in about half of the patients during disease. Management relies on careful follow-up of symptoms and diet. No common agreement was reached on sugar restriction and treatment in adulthood. We administered two dietary regimens differing in their protein and carbohydrate content, high-protein (HPD) and high-protein/glucose-free (GFD), to our mouse model of GSDIII, starting at one month of age. Mice were monitored, either by histological, biochemical and molecular analysis and motor functional tests, until 10 months of age. GFD ameliorated muscle performance up to 10 months of age, while HPD showed little improvement only in young mice. In GFD mice, a decreased muscle glycogen content and fiber vacuolization was observed, even in aged animals indicating a protective role of proteins against skeletal muscle degeneration, at least in some districts. Hepatomegaly was reduced by about 20%. Moreover, the long-term administration of GFD did not worsen serum parameters even after eight months of high-protein diet. A decreased phosphofructokinase and pyruvate kinase activities and an increased expression of Krebs cycle and gluconeogenesis genes were seen in the liver of GFD fed mice. Our data show that the concurrent use of proteins and a strictly controlled glucose supply could reduce muscle wasting, and indicate a better metabolic control in mice with a glucose-free/high-protein diet

Glycogen storage disease type III : A novel Agl knockout mouse model

Glycogen storage disease type III is an autosomal recessive disease characterized by a deficiency in the glycogen debranching enzyme, encoded by AGL. Essential features of this disease are hepatomegaly, hypoglycemia, hyperlipidemia, and growth retardation. Progressive skeletal myopathy, neuropathy, and/or cardiomyopathy become prominent in adults. Currently, there is no available cure. We generated an Agl knockout mouse model by deletion of the carboxy terminus of the protein, including the carboxy end of the glucosidase domain and the glycogen-binding domain. Agl knockout mice presented serious hepatomegaly, but we did not observe signs of cirrhosis or adenomas. In affected tissues, glycogen storage was higher than in wild-type mice, even in the central nervous system which has never been tested in GSDIII patients. The biochemical findings were in accordance with histological data, which clearly documented tissue impairment due to glycogen accumulation. Indeed, electron microscopy revealed the disruption of contractile units due to glycogen infiltrations. Furthermore, adult Agl knockout animals appeared less prompt to move, and they exhibited kyphosis. Three-mo-old Agl knockout mice could not run, and adult mice showed exercise intolerance. In addition, older affected animals exhibited an accelerated respiratory rate even at basal conditions. This observation was correlated with severe glycogen accumulation in the diaphragm. Diffuse glycogen deposition was observed in the tongues of affected mice. Our results demonstrate that this Agl knockout mouse is a reliable model for human glycogenosis type III, as it recapitulates the essential phenotypic features of the disease

In vitro analysis of splice site mutations in the CLCN1 gene using the minigene assay

Mutations in the chloride channel gene CLCN1 cause the allelic disorders Thomsen (dominant) and Becker (recessive) myotonia congenita (MC). The encoded protein, ClC-1, is the primary channel that mediates chloride (Cl-) conductance in skeletal muscle. Mutations in CLCN1 lower the channel's threshold voltage, leading to spontaneous action potentials that are not coupled to neuromuscular transmission and resulting in myotonia. Over 120 mutations in CLCN1 have been described, 10\ua0% of which are splicing defects. Biological specimens suitable for RNA extraction are not always available, but obtaining genomic DNA for analysis is easy and non-invasive. This is the first study to evaluate the pathogenic potential of novel splicing mutations using the minigene approach, which is based on genomic DNA analysis. Splicing mutations accounted for 23\ua0% of all pathogenic variants in our cohort of MC patients. Four were heterozygous mutations in four unrelated individuals, belonging to this cohort: c.563G>T in exon 5; c.1169-5T>G in intron 10; c.1251+1G>A in intron 11, and c.1931-2A>G in intron 16. These variants were expressed in HEK 293 cells, and aberrant splicing was verified by in vitro transcription and sequencing of the cDNA. Our findings confirm the need to further investigate the nature of rearrangements associated with this class of mutations and their effects on mature transcripts. In particular, splicing mutations predicted to generate in-frame transcripts may generate out-of-frame mRNA transcripts that do not produce functional ClC-1. Clinically, incomplete molecular evaluation could lead to delayed or faulty diagnosis. \ua9 2014 Springer Science+Business Media Dordrecht

Impact of a seabird-breeding colony on the vegetation of a small Mediterranean island

To asses the impact of seabirds on the vegetation of a Mediterranean islet, we compared the plant community of Bergeggi islet, harbouring the largest colony of yellow-legged gulls od Liguria (NW Italy

Morpholino antisense oligomer against SOD1 for amyotrophic lateral sclerosis therapy

Neurotoxicity from accumulation of misfolded/mutant proteins is thought to drive pathogenesis in neurodegenerative diseases. Mutations in superoxide dismutase 1 (SOD1) are linked to familial amyotrophic lateral sclerosis (FALS) resulting in progressive motor neuron death through one or more acquired toxicities. Interestingly wild-type SOD1 has been associated also to sporadic ALS (SALS), as misfolded SOD1 has been reported in affected tissues of sporadic patients. For these reasons, it represents a promising therapeutic target for antisense oligonucleotides. We now report slowed disease progression, ameliorated neuromuscular function and increased survival in an ALS in vivo model following therapeutic delivery of morpholino (MO) oligonucleotides designed to reduce the synthesis of ALS-causing human SOD1. Neuropathological analysis demonstrated an increased motor neuron and axon number, an ameliorated muscle trophism and a reduced micro and macrogliosis-mediated inflammation. Moreover, MO yield robust SOD1 suppression, in particular of misfolded form, not only in vivo in ALS rodent, but also in human patient samples, setting the stage for MO-mediated therapy in human clinical trials