Sabrina Lucchiari ^1,2* Francesco Fortunato ² Giovanni Meola ³ Andrea Mignarri ⁴ Serena Pagliarani ⁵ Stefania Corti ² Giacomo Pietro Comi ² Dario Ronchi ²

• ¹ University of Milan, Milan, Italy
• ² Dino Ferrari Centre, Department of Neurological Sciences, University of Milan, IRCCS Fondazione Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy, Milano, Italy
• ³ Department of Neurorehabilitation Sciences, Casa di Cura Igea, University of Milan, Fondazione Malattie Miotoniche, Milan, Lombardy, Italy
• ⁴ Unit of Neurology and Neurometabolic Diseases, Department of Medical, Surgical and Neurological Sciences, University of Siena, Siena, Italy, Siena, Tuscany, Italy
• ⁵ IRCCS Ca 'Granda Foundation Maggiore Policlinico Hospital, Milan, Lombardy, Italy

Myotonia congenita, both in a dominant (Thomsen disease) and in a recessive form (Becker disease), is caused by molecular defects in CLCN1 that encodes the major skeletal muscle chloride channel ClC-1. This channel is important for the normal repolarisation of muscle action potentials and consequent relaxation of the muscle and its dysfunction leads to impaired muscle relaxation after voluntary or evoked contraction and muscle stiffness. More than 300 CLCN1 pathogenic variants have been found in association with congenital myotonia, inherited as recessive or dominant traits (with complete or incomplete penetrance). Herein we describe the case of a 44-year-old woman complaining "leg stiffness" since the age of 20 years and presenting transient muscle weakness especially after seating for several minutes, with grip myotonia and feet myotonia, cold sensitive and warm-up. The strength was normal but muscle hypertrophy at lower limbs was evident. EMG myotonia was detected in all explored muscles. Patient's father had precocious cataract correction but did not show myotonic discharges at EMG. Examination of patient's sons (aged 18 years and 12 years) was unremarkable. The patient started treatment with Mexiletine, with improvement of grip myotonia and limb stiffness but it was soon interrupted for GI disturbances. Direct sequencing of CLCN1 identified the previously described heterozygous intronic variant c.1471+1G>A which resulted in the skipping of Exon 13 in CLCN1 muscle transcript. In addition, the rare heterozygous synonymous nucleotide change c.762C>T p.Cys254Cys was identified and predicted to alter physiological splicing. The detection of multiple splicing abnormalities leading to premature termination codons supported the in-silico prediction. We developed a western blot assay to assess ClC-1 protein in muscle biopsy and we observed that ClC-1 levels were consistently reduced in patient's muscle, supporting the pathogenic behaviour of the variants disclosed. Overall, we report a novel case of Becker myotonia and we highlight the importance of multiple levels of analysis to achieve a firm molecular diagnosis.