REPORT Defective Presynaptic Choline Transport Underlies Hereditary Motor Neuropathy Katy E.S. Barwick, 1,8 Jane Wright, 2,8 Saeed Al-Turki, 3,8 Meriel M. McEntagart, 4,8 Ajith Nair, 1 Barry Chioza, 1 Ali Al-Memar, 5 Hamid Modarres, 5 Mary M. Reilly, 6 Katherine J. Dick, 1 Alicia M. Ruggiero, 2 Randy D. Blakely, 2,7 Matt E. Hurles, 3 and Andrew H. Crosby 1, * The neuromuscular junction (NMJ) is a specialized synapse with a complex molecular architecture that provides for reliable transmission between the nerve terminal and muscle fiber. Using linkage analysis and whole-exome sequencing of DNA samples from subjects with distal hereditary motor neuropathy type VII, we identified a mutation in SLC5A7, which encodes the presynaptic choline transporter (CHT), a critical determinant of synaptic acetylcholine synthesis and release at the NMJ. This dominantly segregating SLC5A7 mutation truncates the encoded product just beyond the final transmembrane domain, eliminating cytosolic-C-terminus sequences known to regulate surface transporter trafficking. Choline-transport assays in both transfected cells and monocytes from affected individuals revealed significant reductions in hemicholinium-3-sensitive choline uptake, a finding consistent with a dominant-negative mode of action. The discovery of CHT dysfunction underlying motor neuropathy identifies a biological basis for this group of conditions and widens the spectrum of disorders that derive from impaired NMJ transmission. Our findings compel consideration of mutations in SLC5A7 or its functional partners in relation to unexplained motor neuronopathies. The distal hereditary motor neuronopathies (dHMNs) comprise a heterogeneous group of diseases that share a common feature of a length-dependent neuropathy re- sulting in progressive distal muscle wasting and weakness. Electrophysiology and electromyography (EMG) studies have revealed normal motor and sensory conduction velocities with reduced compound-motor-action-potential amplitudes and neurogenic changes, suggesting that pathology is at the level of the anterior horn cell. Harding described seven subtypes of dHMNs on the basis of clinical and genetic criteria. 1 Isolation of the causative genes in the dominant forms of dHMNs has revealed genetic heteroge- neity within subtypes, and mutations in six genes have been described to date and include DCTN1 (MIM 601143), GARS (MIM 600287), BSCL2 (MIM 606168), HSPB8 (MIM 608041), HSPB1 (MIM 602195), and HSPB3 (MIM 604624). These findings implicate processes such as protein misfolding, RNA metabolism, and axonal trans- port as pathologic mechanisms in this disorder. 2 Several of these genes also cause forms of Charcot-Marie-Tooth disease type 2 (CMT2), which is categorized separately on the basis of sensory-nerve involvement. We previously detailed the clinical features of members of a large UK family affected by dominantly transmitted dHMN type VII (dHMN-VII [MIM 158580]), distinguished by the presence of vocal-cord involvement in affected subjects, and we mapped the gene responsible to chromo- somal region 2q14. 3–5 Here, we report the identification of a mutation within SLC5A7 (MIM 608761) as the under- lying cause of dHMN-VII. SLC5A7 encodes the presynaptic choline transporter (CHT), a critical determinant of synaptic acetylcholine (ACh) synthesis and release at the neuromuscular junction (NMJ). Mutations affecting genes coding for NMJ proteins are well known to cause a contrast- ing phenotype of congenital myasthenic syndrome, which is characterized by fatiguing muscle weakness and cranial- nerve and respiratory-muscle involvement. 6 Our findings are thus unexpected and widen the phenotypical spectrum associated with NMJ dysfunction. Our studies derive from the analysis of samples from 26 family members (14 affected and 12 unaffected) obtained with informed consent; the research protocol was approved by the Wandsworth Local Research Ethics Committee. In order to identify the disease-associated gene, we performed whole-exome sequencing of a single affected individual (VI:5) in this family (Figure 1). Coding regions were captured with SureSelect All Exons (50 Mb) and sequenced by Illumina HiSeq, yielding 9.8 Gb data (~130 million reads) corresponding to 91% target coverage with a mean depth of 1073 and identifying 52,806 vari- ants (Tables S2 and S3, available online). After filtering, only one probably deleterious variant was identified within the critical region; this was a single base deletion (c.1497delG) in SLC5A7 (RefSeq accession number NM_021815.2), encoding the Na þ /Cl À dependent, high- affinity CHT 7–9 (Figure S1). Dideoxy sequence analysis, as well as Ssp1 restriction digestion (Figure 1), confirmed cosegregation of the variant with the disease phenotype, 1 Centre for Human Genetics, St. George’s University of London, Cranmer Terrace, London SW17 0RE, UK; 2 Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232-6600, USA; 3 WellcomeTrust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK; 4 Medical Genetics, Clinical Developmental Sciences, St. George’s University of London, Cranmer Terrace, London SW17 0RE, UK; 5 Department of Neurology, Atkinson Morley Wing, St. George’s Hospital, Tooting, London SW17 0RE, UK; 6 Medical Research Council Centre for Neuromuscular Diseases, University College London Institute of Neurology, Queen Square, London WC1N 3BG, UK; 7 Department of Psychiatry, Vanderbilt University School of Medicine, Nashville, TN 37212, USA 8 These authors contributed equally to this work *Correspondence: acrosby@sgul.ac.uk http://dx.doi.org/10.1016/j.ajhg.2012.09.019. Ó2012 by The American Society of Human Genetics. All rights reserved. The American Journal of Human Genetics 91, 1103–1107, December 7, 2012 1103