ARTICLE Mutations in ANO3 Cause Dominant Craniocervical Dystonia: Ion Channel Implicated in Pathogenesis Gavin Charlesworth, 1 Vincent Plagnol, 2 Kira M. Holmstro ¨m, 1 Jose Bras, 1 Una-Marie Sheerin, 1 Elisavet Preza, 1 Ignacio Rubio-Agusti, 3,4 Mina Ryten, 1,8 Susanne A. Schneider, 5 Maria Stamelou, 3 Daniah Trabzuni, 1,6,8 Andrey Y. Abramov, 1 Kailash P. Bhatia, 3,7, * and Nicholas W. Wood 1,2,7, * In this study, we combined linkage analysis with whole-exome sequencing of two individuals to identify candidate causal variants in a moderately-sized UK kindred exhibiting autosomal-dominant inheritance of craniocervical dystonia. Subsequent screening of these candidate causal variants in a large number of familial and sporadic cases of cervical dystonia led to the identification of a total of six putatively pathogenic mutations in ANO3, a gene encoding a predicted Ca 2þ -gated chloride channel that we show to be highly ex- pressed in the striatum. Functional studies using Ca 2þ imaging in case and control fibroblasts demonstrated clear abnormalities in endo- plasmic-reticulum-dependent Ca 2þ signaling. We conclude that mutations in ANO3 are a cause of autosomal-dominant craniocervical dystonia. The locus DYT23 has been reserved as a synonym for this gene. The implication of an ion channel in the pathogenesis of dys- tonia provides insights into an alternative mechanism that opens fresh avenues for further research. Introduction Cervical dystonia is the most common form of focal dysto- nia seen by neurologists. 1 Previous epidemiological studies conducted in Europe and Northern England have sug- gested a prevalence of 5.7–6.1 per 100,000 persons. 2,3 Although lifespan is not generally reduced, individuals affected by the condition can suffer considerable physical and psychosocial distress, which has been shown to have a significant impact on their quality of life. 4 Treatment remains symptomatic, and regular injections of botulinum toxin constitute the mainstay of current medical therapy. Genetic factors are believed to play an important role in the pathogenesis of cervical dystonia given that around 10%–20% of affected individuals have one or more affected family members. 5,6 Despite this fact, a later age of onset and characteristically reduced penetrance have made it difficult to identify kindreds of a size sufficient to permit traditional linkage-based approaches to gene identifica- tion. So far, mutations in two genes (TOR1A [MIM 605204] and THAP1 [MIM 609520]) have been conclu- sively shown to cause autosomal-dominant primary dysto- nia. 7,8 Even together, however, mutations in these genes explain only a small fraction of familial dystonia, suggest- ing that a number of genetic factors remain to be identi- fied. More recently, mutations is CIZ1 (MIM 611420) have also been suggested as a cause of adult-onset cervical dystonia, 9 although this has yet to be confirmed by others. In this study, we combined linkage analysis with whole- exome sequencing of two individuals to identify candidate causal variants in a moderately-sized UK kindred exhibit- ing autosomal-dominant inheritance of primary craniocer- vical dystonia 10 (TOR1A and THAP1 had previously been excluded). We performed Sanger sequencing of the candi- date variant in a large number of dystonia samples and subsequent next-generation targeted sequencing of the whole gene to provide a comprehensive genetic screening of phenotypically similar cases. Subjects and Methods The Index Family All samples were collected with the written consent of participants and formal ethical approval by the relevant research ethics committee. All living individuals from the index family were re- examined and videoed as part of this study, and the two now deceased individuals had been examined and videoed as part of a previous study. 10 The family pedigree is shown in Figure 1. All family members shown are over 25 years of age. Upon examina- tion, all definitely affected family members exhibited tremulous cervical dystonia with a variable degree of associated upper-limb dystonic tremor. In addition, family members II-7 and III-7 had laryngeal involvement, and family member II-4 had both laryngeal involvement and blephrospasm (see Movie S1, available online). Age of onset ranged from 19–39 years, and most had onset in the last few years of their fourth decade. One family member, II-1, had additional neurological signs on examination: he exhibited mild truncal ataxia, dysarthria, and mild cognitive impairment, all dating from an episode of Wernicke-Korsakoff’s encephalopathy 6 years previously. His family confirmed that he had consumed alcohol excessively for much of his life prior to that episode. 1 Department of Molecular Neuroscience, University College London Institute of Neurology, Queen Square, London WC1N 3BG, UK; 2 University College London Genetics Institute, London WC1E 6BT, UK; 3 Sobell Department for Movement Disorders, University College London Institute of Neurology, London WC1N 3BG, UK; 4 Movement Disorders Unit, Hospital Universitario La Fe, 46009 Valencia, Spain; 5 Department of Neurology, University of Kiel, Arnold-Heller-Straße 3, 24105 Kiel, Germany; 6 King Faisal Specialist Hospital and Research Centre, Department of Genetics, PO Box 3354, Riyadh 11211, Saudi Arabia 7 These authors contributed equally to this work 8 On behalf of the UK Human Brain Expression Consortium *Correspondence: n.wood@ucl.ac.uk (N.W.W.), k.bhatia@ucl.ac.uk (K.P.B.) http://dx.doi.org/10.1016/j.ajhg.2012.10.024. Ó2012 by The American Society of Human Genetics. All rights reserved. The American Journal of Human Genetics 91, 1041–1050, December 7, 2012 1041