Movement disorders J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2012-304131 on 24 January 2013. Downloaded from RESEARCH PAPER Clinical, genetic, neurophysiological and functional study of new mutations in episodic ataxia type 1 Susan Elizabeth Tomlinson,1,2,3,4 Sanjeev Rajakulendran,1,2 Stella Veronica Tan,1,2 Tracey Dawn Graves,1,2 Doris-Eva Bamiou,1 Robyn W Labrum,1,2 David Burke,3 Carolyn M Sue,5 Paola Giunti,1,2 Stephanie Schorge,2 Dimitri M Kullmann,1,2 Michael G Hanna1,2 ▸ Additional material is ABSTRACT the expression and localisation of Kv channels, as published online only. To view Background and objective Heterozygous mutations well as their gating and kinetic properties.12 please visit the journal online + (http://dx.doi.org/10.1136/ in KCNA1 cause episodic ataxia type 1 (EA1), an ion Fast K channels that contain Kv1.1 subunits jnnp-2012-304131). channel disorder characterised by brief paroxysms of usually occur as heterotetramers in combination with cerebellar dysfunction and persistent neuromyotonia. This K 1.2 or K 1.4 subunits. These channels are mostly 1MRC Centre for v v Neuromuscular Disease, paper describes four previously unreported families with closed at resting membrane potential and have a rela- National Hospital for EA1, with the aim of understanding the phenotypic tively low threshold for activation and rapid activa- Neurology and Neurosurgery, spectrum associated with different mutations. tion kinetics, but are slower than fast Na+ channels London, UK Methods 15 affected individuals from four families (hence the term ‘delayed rectifier’). K+ ion efflux, 2Institute of Neurology, University College London, underwent clinical, genetic and neurophysiological driven by the electrochemical trans-membrane gradi- London, UK evaluation. The functional impact of new mutations ent, has a hyperpolarising effect on the membrane 3Institute of Clinical identified in the KCNA1 gene was investigated with in potential and therefore limits neuronal excitability.34 Neuroscience, Royal Prince + vitro electrophysiology and immunocytochemistry. In myelinated nerves, Kv1.1-containing K channels Alfred Hospital, University of Results Detailed clinical documentation, dating back to are densely expressed in the juxtaparanodal region of Sydney, Sydney, New South 2 + Wales, Australia 1928 in one family, indicates that all patients manifested the axon. Activation of fast K channels reduces the 4 Department of Neurology, St episodic ataxia of varying severity. Four subjects from resistance of the internodal membrane and limits copyright. Vincent’s Hospital, Sydney, three families reported hearing impairment, which has axonal hyperexcitability after an action potential.56 New South Wales, Australia not previously been reported in association with EA1. Dysfunction is thus expected to lead both to excessive 5Department of Neurology, Royal North Shore Hospital and New mutations (R167M, C185W and I407M) were excitability of neurons and to an enhanced duration Kolling Institute of Medical identified in three out of the four families. When of action potentials, which may in turn cause exces- Research, University of Sydney, expressed in human embryonic kidney cells, all three sive neurotransmitter release. These effects contrib- Sydney, New South Wales, new mutations resulted in a loss of Kv1.1 channel ute to the repetitive activity and paraesthesiae elicited Australia 7 function. The fourth family harboured a previously by the Kv blocker 4-aminopyridine. Of the many + + Correspondence to reported A242P mutation, which has not been other K channels, slow K channels encoded by Professor Dimitri M Kullmann, previously described in association with ataxia. KCNQ genes are especially important in neurological UCL Institute of Neurology, Conclusions The genetic basis of EA1 in four families disease, such as benign familial neonatal epilepsy.8 Queen Square, is established and this report presents the earliest They co-assemble to form outward rectifying cur- http://jnnp.bmj.com/ London WC1N 3BG, UK; [email protected] documented case from 1928. All three new mutations rents that helps stabilise the membrane potential. In caused a loss of Kv1.1 channel function. The finding of neurones, these commonly exist as Kv7.2–Kv7.3 het- Received 24 October 2012 deafness in four individuals raises the possibility of a link eromers, which activate slowly and are non- Revised 18 December 2012 39 + between Kv1.1 dysfunction and hearing impairment. Our inactivating. Slow K channels are highly Accepted 27 December 2012 findings broaden the phenotypic range associated with expressed in myelinated nerve at the nodes of Published Online First 10 11 24 January 2013 mutations in KCNA1. Ranvier and the axonal initial segments, and in the central nervous system they are abundant at the on September 23, 2021 by guest. Protected axonal initial segments.10 Heterozygous mutations in the KCNA1 gene INTRODUCTION (chromosome 12) encoding the α subunit of Kv1.1 12 Voltage-gated potassium channels (Kv channels) result in episodic ataxia type 1. EA1 is a domin- play fundamental roles in controlling neuronal antly inherited condition characterised by brief excitability. The main pore-forming channel is com- attacks of midline cerebellar disturbance including posed of four α subunits, of which approximately limb ataxia, dysarthria, titubation, nystagmus, ▸ 40 subtypes are known, organised in 12 subfamilies tremor and gait incoordination. Episodes may be http://dx.doi.org/10.1136/ – jnnp-2012-304857 (Kv1 Kv12). They include two subgroups relevant triggered by startle, vigorous activity, change in to disorders of peripheral nerve excitability, ataxia posture (eg, from sitting to standing), emotion, and epilepsy: fast potassium channels (Kv1), and hunger, alcohol or intercurrent illness. At the onset slow potassium channels (Kv7). The Kv channel of events, some of our patients describe the sensa- To cite: Tomlinson SE, β ‘ ’ ‘ ’ Rajakulendran S, Tan SV, also contains four intracellular auxiliary subunits. tion of a click or a shock in their head. Episodes et al. J Neurol Neurosurg The specificity of α subunits for β subunits is con- last seconds to minutes and may be associated with Psychiatry 2013;84: ferred by the cytosolic N-terminus of the α a feeling of apprehension or anxiety. Cerebellar – 1107 1112. subunit, and the combination of subunits influences function is typically normal between attacks. Tomlinson SE, et al. J Neurol Neurosurg Psychiatry 2013;84:1107–1112. doi:10.1136/jnnp-2012-304131 1107 Movement disorders J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2012-304131 on 24 January 2013. Downloaded from However, almost all patients exhibit persistent neuromyotonia Electrophysiology which can be confirmed on electromyography (EMG).13 14 An Whole-cell patch clamp recordings were performed to record increased incidence of epilepsy has also been reported in associ- potassium currents at room temperature. The external solution ation with EA1. Brain imaging in patients with EA1 is typically contained (in mM): NaCl2 135; KCl 4; MgCl2 1; CaCl2 2; – unremarkable.15 18 HEPES 10. The pipette solution contained (in mM): NaCl 135; Over 25 mutations have been identified in KCNA1, with func- KCl 2.5; EGTA 2; HEPES 10. Pipette resistance was 2–4MΩ tional studies demonstrating a loss of channel function through when filled with intracellular solution. Cells were held at a dominant-negative mechanism.13 16 However, the full clinical −80 mV, and series resistance was not compensated. Cells with spectrum of EA1 is unknown. Here we describe new clinical series resistance above 10 MΩ were discarded. A –P/4 protocol and genetic findings in four previously unreported families with was used to subtract leak currents. Currents were recorded EA1. In addition to typical features of EA1, several individuals using an Axopatch 200B amplifier (Molecular Devices). Data reported hearing impairment. New heterozygous mutations in were acquired and analysed using LabView software (V.8.0, KCNA1 were identified in three of the four families. All three National Instruments) with in-house acquisition programs new mutations caused a loss of KV1.1 function. The fourth (DMK). Data were sampled at 20 kHz and filtered at 5 kHz. family carried the A242P mutation which has previously been Graphs were plotted using Sigmaplot software (V.8.0, SPSS). reported in association with epilepsy and neuromyotonia but Statistical significance was determined using one-way analysis of not with ataxia.16 variance. METHODS Immunocytochemistry Ethics approval was obtained from the Joint National Hospital HEK cells were co-transfected using lipofectamine with for Neurology and Neurosurgery/University College London pMT2LF KCNA1 and pcDNA3.1 GFP constructs 48 h prior to Ethics Committee and the University of Sydney Research Ethics immunocytochemistry. Cells were initially fixed for 5 min with Committee for this project. All subjects provided written 2% and then 4% of paraformaldehyde, followed by washing informed consent. Patients were assessed clinically and, where with 0.01% phosphate buffered saline solution (PBS). Cells possible, nerve conduction studies and EMG were performed were permeabilised with 0.1% Triton X-100 in PBS for 5 min according to a previously described protocol.13 Non-invasive and then incubated with 4% normal goat serum for 20 min. nerve excitability studies were also performed in this cohort, Cells were incubated with the primary antibody, 1:500 rabbit and have been reported elsewhere.8