Clinical and molecular study of a new form of hereditary myotonia in Murrah water buffalo Alexandre S. Borges a,⇑ , Jose ´ D. Barbosa b , Luiz Anto ˆnio L. Resende c , Lı ´gia S.L.S. Mota d , Roge ´rio M. Amorim a , Thaı ´s L. Carvalho d , Jose ´ F. Garcia e , Jose ´ P. Oliveira-Filho a , Carlos M.C. Oliveira b , Jorge Estefano S. Souza f , Nena J. Winand g a Department of Veterinary Clinical Science, College of Veterinary Medicine and Animal Science, Univ Estadual Paulista (UNESP), Botucatu, Brazil b Faculdade de Veterina ´ ria, Universidade Federal do Para ´ , Castanhal, Brazil c Department of Neurology, Faculty of Medicine, Univ Estadual Paulista (UNESP), Botucatu, Brazil d Department of Genetics, Biosciences Institute, Univ Estadual Paulista (UNESP), Botucatu, Brazil e Departamento de Apoio, Produc ßa ˜o e Sau ´ de Animal, Univ Estadual Paulista (UNESP), Arac ßatuba, Brazil f Bioinformation J2ss Informa ´ tica Ltda ME Sao Paulo, Brazil g Department of Molecular Medicine, Cornell University, Ithaca, NY, USA Received 5 May 2012; received in revised form 4 November 2012; accepted 12 November 2012 Abstract Hereditary myotonia caused by mutations in CLCN1 has been previously described in humans, goats, dogs, mice and horses. The goal of this study was to characterize the clinical, morphological and genetic features of hereditary myotonia in Murrah buffalo. Clinical and laboratory evaluations were performed on affected and normal animals. CLCN1 cDNA and the relevant genomic region from normal and affected animals were sequenced. The affected animals exhibited muscle hypertrophy and stiffness. Myotonic discharges were observed during EMG, and dystrophic changes were not present in skeletal muscle biopsies; the last 43 nucleotides of exon-3 of the CLCN1 mRNA were deleted. Cloning of the genomic fragment revealed that the exclusion of this exonic sequence was caused by aberrant splicing, which was associated with the presence of a synonymous SNP in exon-3 (c.396C>T). The mutant allele triggered the efficient use of an ectopic 5’ splice donor site located at nucleotides 90–91 of exon-3. The predicted impact of this aberrant splicing event is the alteration of the CLCN1 translational reading frame, which results in the incorporation of 24 unrelated amino acids followed by a premature stop codon. Ó 2012 Elsevier B.V. All rights reserved. Keywords: Hereditary myotonia; Chloride channel; Cryptic splicing; Water buffalo 1. Introduction Myotonia is the phenomenon of delayed muscle relaxation after contraction [1]. Myotonia congenita is an inherited disorder of muscle membrane hyperexcitability caused by reduced sarcolemmal chloride conductance due to mutations in the skeletal muscle voltage-gated chloride channel gene (CLCN1), which encodes the main skeletal muscle chloride channel (CLC1) [2]. This disorder is the most common heritable skeletal muscle ion channelopathy in humans [3]. Nearly 30 years after the first description of myotonia in humans, a clinically similar disease was described in goats [4]. Studies performed on myotonic goats were initially important for delineating the physiological basis of myotonia and demonstrating the abnormal excitability of the muscle [5]. Subsequent studies 0960-8966/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.nmd.2012.11.008 ⇑ Corresponding author. Address: Department of Veterinary Clinical Science, College of Veterinary Medicine and Animal Science, Univ Estadual Paulista (UNESP), Botucatu, SP 18618970, Brazil. Tel./fax: +55 1438802049. E-mail address: asborges@fmvz.unesp.br (A.S. Borges). www.elsevier.com/locate/nmd Available online at www.sciencedirect.com Neuromuscular Disorders 23 (2013) 206–213