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’s disease) or recessive manner (Becker’s 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.