SHORT REPORTS EFFECTS OF COIL CHARACTERISTICS FOR FEMORAL NERVE MAGNETIC STIMULATION KATJA TOMAZIN, PhD,1 SAMUEL VERGES, PhD,2 NICOLAS DECORTE, MSc,2 ALAIN OULERICH, BSc,1 and GUILLAUME Y. MILLET, PhD1 1 2 Exercise Physiology Laboratory, Jean Monnet University, Saint-Etienne, France REX-S Laboratory, Joseph Fourier University and Exercise Research Unit, Grenoble, University Hospital, Grenoble, France Accepted 16 September 2009 ABSTRACT: The aim of this study was to compare the efficiency of two coils used for femoral nerve magnetic stimulation and to compare them with electrical stimulation in inducing maximal response of the quadriceps. The mechanical and electromyographic (EMG) responses were dependent on the coil used. The 45-mm double coil showed greater efficiency to elicit a maximal quadriceps response, which was similar to electrical stimulation. Muscle Nerve 41: 406–409, 2010 Femoral nerve magnetic stimulation (FNMS) is a technique used to assess neuromuscular function that was developed several years ago1 but has been popularized recently.2–4 Laghi5 suggested that this technique could have important clinical implications. To be reliable and allow quantification of the muscle vs. central components of neuromuscular function, stimulation of the femoral nerve must be supramaximal; that is, a further increase in stimulus intensity should not increase the mechanical and electromyographic (EMG) quadriceps responses. For instance, it has been suggested that obesity could affect the capacity of magnetic stimulation to obtain supramaximal stimuli,5 but the characteristics of the magnetic stimulation device may also alter the response. We recently validated the technique in comparison with electrically evoked stimulation6 by using a Magstim 200 device (Magstim Co., Whiteland, Dyfed, UK) connected to a 45mm double (or figure-of-eight) coil (2.5 T). Currently, a new second-generation double 70-mm coil is available (Magstim Co., Whiteland, Dyfed, UK) for FNMS, and similar coils have been used in recent studies.2,7,8 Because the rise time and peak magnetic field transferred to the coil depends not only on the characteristics of the stimulator, but also on the characteristics of the coil, the neuromuscular response produced by a new second-generation coil should be evaluated. Thus, the aim of our study was to compare the magnitude of the isometAbbreviations: ANOVA, analysis of variance; EMG, electromyography; FNES, femoral nerve electrical stimulation; FNMS, femoral nerve magnetic stimulation; PPA, peak-to-peak M-wave amplitude; TWP, twitch peak torque Key words: electrical stimulation; femoral nerve; isometric twitch; magnetic stimulation; M-wave Correspondence to: G.Y. MILLET; e-mail: guillaume.millet@univ-stetienne.fr C 2009 Wiley Periodicals, Inc. V Published online 10 November 2009 in Wiley InterScience (www. interscience.wiley.com). DOI 10.1002/mus.21566 406 Effects of Magnetic Coil Characteristics ric twitch and M-wave response of quadriceps muscle elicited at given intensities with a validated6 45mm double coil vs. a new second-generation double 70-mm coil. Furthermore, we aimed to evaluate the evoked quadriceps response with both coils at 100% stimulation intensity vs. the evoked response elicited with widely used standard femoral nerve electrical stimulation, place parentheses around (FNES).6 METHODS Nine healthy male subjects were studied (weight 71.8 6 7.9 kg, height 177.7 6 5.5 cm, age 31.3 6 6.9 years, body mass index 22.8 6 2.9 kg/ m2). Written informed consent was obtained from all subjects. The study was conducted according to the Declaration of Helsinki. Subjects. Subjects lay supine on a table with the knee-joint angle set at 90 . The distal part of the right shank was connected with a non-elastic strap to a force transducer (SBB 200 kg; Tempo Technologies, Taipei, Taiwan) just above the tip of the lateral malleolus. The subjects lay on the table for 10 min before the coil was placed high in the femoral triangle just lateral to the femoral artery. To determine the best coil position to produce the maximal quadriceps response, the coil was placed 3–5 cm below the inguinal ligament and parallel to its course (see Fig. 1 in Verges et al.6). FNMS were performed with two coils: (1) a 45-mm double coil (2.5 T) and (2) a double 70mm coil (2.2 T). Five stimulation intensities (100%, 95%, 90%, 85%, and 80% of maximum power output of the Magstim 2002; Magstim Co., Whiteland, Dyfed, UK), and the two coils were used in random order. The stimulation duration was 0.1 ms. Two isometric twitches were evoked at the same intensity with a 20-s rest interval between stimulations. In all subjects, FNES was performed with the cathode electrode (10-mm diameter; Ag–AgCl, Type 0601000402; Contrôle Graphique Medical, Brie-Comte-Robert, France) pressed over the femoral nerve in the femoral triangle, 3–5 cm below the inguinal ligament and the anode (10.2 cm  5.2 cm; Compex, SA, Ecublens, Switzerland) placed over the gluteal fold. Electrical impulses (single, square-wave, 1-ms duration) were delivered by a constant-current, high-voltage (maximal voltage Procedures. MUSCLE & NERVE March 2010 400 V) stimulator (DS7A; Digitimer, Hertfordshire, UK). The surface EMG signal was recorded from the right vastus lateralis (VL) muscle with two pairs of bipolar oval self-adhesive electrodes with 2.5-cm interelectrode distance (10-mm diameter; Ag– AgCl, Type 0601000402; Contrôle Graphique Medical, Brie-Comte-Robert, France). The position and placement of the electrodes were made according to the Surface Electromyography for the Non-Invasive Assessment of Muscles project (SENIAM) recommendations. EMG data were recorded (PowerLab System 16/30, ML880/P; ADInstruments, Sydney, Australia) at a sampling frequency of 2000 HZ. EMG signals were amplified with an octal bioamplifier (ML138; ADInstruments) with a bandwidth frequency ranging from 3 HZ to 1 kHZ (input impedance ¼ 200 MX, common mode rejection ratio ¼ 85 dB, gain ¼ 1000), transmitted to the PC and analyzed with LabChart5 software (ADInstruments). Data Analysis. Quadriceps muscle isometric twitch peak torque (TWP) and VL M-wave peak-to-peak amplitude (PPA) were analyzed for each condition. Relative changes in TWP and PPA were calculated according to the values obtained at 100% of maximal power output with the 45-mm double coil. Statistical Analysis. Data are presented as mean (6 SD) in the text and as mean (6 SE) in Figure 1. To detect significant differences between FNMS at 100% of maximum stimulator power output with both coils and FNES, a one-way analysis of variance (ANOVA) for repeated measures with one withinsubject factor was used. To detect significant differences between coils, a two-way ANOVA for repeated measures with two within-subjects factors (stimulation intensity  coil) was used. When significant main effects were found, Tukey’s post hoc test was used to identify the location of the differences. Standard statistical software (Statistica 6.0; StatSoft, Inc., Tulsa, Oklahoma) was used. The significance level for all comparisons was set at P < 0.05. RESULTS The mean TWP evoked with the 45-mm coil was higher compared with mean values evoked with the 70-mm coil at all intensities of the stimulator output (Fig. 1), because ANOVA revealed a significant main effect of coil (P < 0.01), intensity (P < 0.001), and coil  intensity interaction (P < 0.001). TWP elicited with the 45-mm coil did not decrease at any intensity level of the stimulator power output; meanwhile, TWP elicited with the 70-mm coil decreased from 85% of maximum stimulator output (Fig. 1). The mean TWP elicited Effects of Magnetic Coil Characteristics with both coils at 100% of maximum stimulator power output was not significantly different from mean TWP values evoked with FNES (Fig. 1). ANOVA also revealed a significant main effect of coil (P < 0.05), intensity (P < 0.001), and interaction between them (P < 0.001) on mean PPA. Mean PPA amplitudes evoked with the 45-mm coil were higher compared with mean values evoked with the 70-mm coil at intensities below 95%, although the M-wave amplitude showed a strong tendency to be higher ( 9.2 6 13.5%) also at 100% of stimulator output (Fig. 1). PPA elicited with the smaller coil did not decrease at any intensity level of the stimulator power output, whereas PPA elicited with the larger coil decreased from an initial value at 85% of maximum stimulator output. The mean PPA evoked with the 45-mm coil at 100% of maximum stimulator power output was not significantly different from the mean PPA evoked with FNES, whereas PPA evoked with the 70-mm coil was 13.7 6 13.2% lower compared with FNES (P ¼ 0.01). DISCUSSION The main goal of this study was to determine whether the magnitude of the elicited quadriceps response could be affected by two different coils designed to perform FNMS. The results show that the mechanical and EMG responses in the quadriceps muscle were dependent on the coil used (Fig. 1). Moreover, the magnitude of the evoked quadriceps response with the 45-mm coil was not different compared with the widely used femoral nerve stimulation, although the same could not be shown with the 70-mm coil according to lower PPA. Because supramaximal stimulation cannot be ensured with the 70-mm coil, particular attention should be made to this point in future studies, especially during fatigue experiments, when excitability of the nerve decreases. Smaller isometric twitches and M-wave amplitudes mean values evoked with the double 70-mm coil could be due to a different coil design, structure, and size.9 For instance, the plastic enclosure wrapped around the 70-mm coil could increase the distance from the discharge magnetic field to the femoral nerve when compared with the 45-mm double coil, which is covered only by a polyurethane coat. Because the power of the magnetic field falls off by approximately 50% at 10 mm from the coil,10 this could be a major reason for the results shown in Figure 1, although the coil with the greater diameter (70 mm) should provide deeper penetration of the magnetic fields than one with a smaller diameter (45 mm).11 Two other reasons for the lower quadriceps mean values of TWP and PPA (Fig. 1) evoked with the 70-mm coil MUSCLE & NERVE March 2010 407 FIGURE 1. Mean (6SE) values of the twitch peak torque (TWP) (A) and peak-to-peak M-wave amplitude (PPA) (B) evoked with the 45-mm double coil (filled squares) and with the 70-mm double coil (open squares) at different percentages of the stimulator’s maximal power output. Values are expressed as relative changes in percent of responses evoked with the 45-mm coil at 100% of the stimulator’s maximal power output. Significant differences between the two coils for TWP and PPA values at: *P < 0.05, **P < 0.01, and ***P < 0.001, respectively; ***significant difference from the initial values evoked at 100% of stimulation intensity for TWP and PPA values evoked with the same coil (P < 0.001); and #significant difference between 70-mm double coil and femoral nerve electric stimulation (FNES). could be: (i) the peak magnetic field intensity (2.5 T for 45 mm vs. 2.2 T for 70 mm), and (ii) the ability of the coil with the smaller diameter to induce a higher magnetic field at shorter distances.11 It seems that, for femoral nerve magnetic stimulation, it is crucial to have a high magnetic field concentrated into a small area, such as with the 45-mm double coil, rather than a smaller field strength spread over longer distances, as produced by the 70-mm coil. In conclusion, this study has confirmed that FNMS and FNES provide similar mechanical and 408 Effects of Magnetic Coil Characteristics EMG responses in the quadriceps muscle6 when a 45-mm coil is used. A 45-mm double coil, which is covered only by a polyurethane coat and is capable of inducing a peak magnetic field up to 2.5 T, is a better tool for stimulating the femoral nerve supramaximally when compared with the new secondgeneration double 70-mm coil. More generally, we conclude that the coil characteristics can severely affect the capacity of magnetic stimulation to induce supramaximal stimuli of the femoral nerve. This may be particularly crucial when evaluating overweight or obese subjects and during fatigue MUSCLE & NERVE March 2010 studies. Future studies should aim to define the ‘‘gold standard’’ for this technique. REFERENCES 1. Polkey MI, Kyroussis D, Hamnegard CH, Mills GH, Green M, Moxham J. Quadriceps strength and fatigue assessed by magnetic stimulation of the femoral nerve in man. Muscle Nerve 1996;19:549–555. 2. Katayama K, Amann M, Pegelow DF, Jacques AJ, Dempsey JA. Effect of arterial oxygenation on quadriceps fatigability during isolated muscle exercise. Am J Physiol Regul Integr Comp Physiol 2007;292: R1279–R1286. 3. Kremenic IJ, Glace BW, Ben-Avi SS, Nicholas SJ, McHugh MP. Central fatigue after cycling evaluated using peripheral magnetic stimulation. Med Sci Sports Exerc 2009;41:1461–1466. 4. Romer LM, Haverkamp HC, Lovering AT, Pegelow DF, Dempsey JA. Effect of exercise-induced arterial hypoxemia on quadriceps muscle fatigue in healthy humans. Am J Physiol Regul Integr Comp Physiol 2006;290:R365–R375. 5. Laghi F. Advancing femoral nerve stimulation into the stage of science. J Appl Physiol 2009;106:356–357. 6. Verges S, Maffiuletti NA, Kerherve H, Decorte N, Wuyam B, Millet GY. Comparison of electrical and magnetic stimulations to assess quadriceps muscle function. J Appl Physiol 2009;106:356–357. 7. O’Brien TD, Reeves ND, Baltzopoulos V, Jones DA, Maganaris CN. Assessment of voluntary muscle activation using magnetic stimulation. Eur J Appl Physiol 2008;104:49–55. 8. Swallow EB, Gosker HR, Ward KA, Moore AJ, Dayer MJ, Hopkinson NS, et al. A novel technique for nonvolitional assessment of quadriceps muscle endurance in humans. J Appl Physiol 2007;103:739–746. 9. Madariaga VB, Manterola AG, Miró EL, Iturri JB. Magnetic stimulation of the quadriceps: analysis of 2 stimulators used for diagnostic and therapeutic applications. Arch Bronconeumol 2007;43:411–417. 10. Jalinous R. Technical and practical aspects of magnetic nerve stimulation. J Clin Neurophysiol 1991;8:10–25. 11. Lin VW, Deng X, Lee YS, Hsiao IN. Stimulation of the expiratory muscles using microstimulators. IEEE Trans Neural Syst Rehabil Eng 2008;4:416–420. SMALL FIBER NEUROPATHY IN FEMALE PATIENTS WITH FABRY DISEASE ROCCO LIGUORI, MD,1 VITANTONIO DI STASI, MD,1 ENRICO BUGIARDINI, MD,1 RENZO MIGNANI, MD,2 ALESSANDRO BURLINA, MD,3 WALTER BORSINI, MD,4 AGOSTINO BARUZZI, MD,1 PASQUALE MONTAGNA, MD,1 and VINCENZO DONADIO, MD1 1 Dipartimento di Scienze Neurologiche, Università di Bologna, Via Ugo Foscolo 7, 40123 Bologna, Italy Department of Nephrology, Degli Infermi Hospital, Rimini, Italy 3 Department of Neurology, S. Bassiano Hospital, Bassano del Grappa (VI), Bassano, Italy 4 Department of Neurology, Careggi Hospital, Florence, Italy 2 Accepted 26 October 2009 ABSTRACT: Recent studies suggest that heterozygous female Fabry disease (FD) patients develop peripheral neuropathy. We used skin biopsy to define somatic and autonomic peripheral nerve characteristics in 21 females with FD who were mainly asymptomatic and had normal renal function. Somatic epidermal and dermal autonomic nerve fiber reductions were found, prevalently in the leg, and no differences were found between symptomatic and asymptomatic individuals. Our findings suggest that females with FD, although asymptomatic, may have somatic and autonomic small fiber neuropathy. Muscle Nerve 41: 409–412, 2010 Fabry disease (FD) is a rare X-linked lysosomal storage disease caused by deficiency of alpha-galactosidase A activity. It usually affects males.1 Recent studies have reported that heterozygous females develop typical FD symptoms including peripheral neuropathy,2 although important aspects, such as early involvement in asymptomatic patients and the type of neuropathy (i.e., length-dependent or autonomic neuropathy or ganglionopathy) were not taken into account. We describe a large cohort of mainly asymptomatic female patients without renal insufficiency who exhibited previously undescribed features of peripheral nerve involvement. Abbreviations: AVAs, arteriovenous anastomoses; ENFD, epidermal nerve fiber density; FD, Fabry disease; MEP, muscle erector pilorum; N, normal control values; SG, sweat glands Key words: small fiber neuropathy; skin biopsy; autonomic dysfunction; Fabry disease; female Correspondence to: R. Liguori; e-mail: rocco.liguori@unibo.it C 2009 Wiley Periodicals, Inc. V Published online 17 December 2009 in Wiley InterScience (www. interscience.wiley.com). DOI 10.1002/mus.21606 Small Fiber Neuropathy in FD MATERIALS AND METHODS We studied 21 untreated female patients (mean age 35 years; range 20–62 years) with FD confirmed by mutation analysis. Fifteen patients were asymptomatic, whereas six reported episodic burning pain in the distal extremities evoked by exercise and/or fever. Two of them complained of loss of sweating in the lower limbs. Renal function (glomerular filtration rate and creatinine clearance) was normal in all patients, and other systemic diseases associated with peripheral neuropathy were absent. This study was approved by the Human Ethics Committee of Bologna University, and all subjects gave their written informed consent. To visualize somatic and autonomic unmyelinated skin fibers, 3-mm punch biopsies were taken from fingertip, distal leg, and thigh. Specimens were fixed, sectioned, and incubated with primary antibodies, mainly to the pan-neuronal marker protein gene product 9.5, collagen IV,3 and to the autonomic fiber marker vasoactive intestinal peptide colocalized in sudomotor cholinergic fibers,4 and dopamine b hydroxylase as a marker for noradrenergic fibers.5 A biotinylated endothelium binding lectin, ULEX europæus (Vector Laboratories, Burlingame, California) was added along with primary antibodies to disclose the endothelium, sweat gland tubules, and hair follicles. Digital images were acquired and studied using a laserscanning confocal microscope (Leica DMIRE 2, Skin Biopsy. MUSCLE & NERVE March 2010 409 TCS SL, Leica Microsystems, Heidelberg, Germany). Epidermal nerve fiber density (ENFD: number of unmyelinated fibers per linear millimeter of epidermis) was calculated by considering single epidermal nerve fiber crossings of the dermal–epidermal junction. Autonomic innervation of skin annexes was graded by a semiquantitative scale.6 It included: 0 ¼ absent autonomic innervation; 1 ¼ severe loss of the fiber amount and/or a destroyed pattern of innervation with fibers showing morphological abnormalities such as swelling and fragmentation; 2 ¼ discrete loss of autonomic fibers showing no or sparse morphological changes with a recognizable but abnormal pattern of innervation; 3 ¼ slightly reduced autonomic fiber density without morphological abnormalities and preserved pattern of innervation; 4 ¼ full nerve fiber density with preserved pattern of innervation. The autonomic innervation score in each skin site represented the mean of three different target structures: sweat glands (SG) for the cholinergic score in both glabrous and hairy skin; arteriovenous anastomoses (AVAs) for adrenergic innervation in glabrous skin; and muscle erector pilorum (MEP) for the adrenergic score in hairy skin. As a measure of internal consistency and reliability, we evaluated intraobserver (V.Don.) and interobserver (V.Don. and E.B.) autonomic innervation variability by blinded comparison. The epidermal and dermal skin innervation from patients were compared with data obtained in 20 age-matched healthy subjects without signs of neurological dysfunction. Skin innervation between patients and controls was analyzed using t-tests for unpaired data, whereas proximal and distal skin innervation in patients was compared by paired t-test. Analyzed data were normally distributed according to the Kolmogorov–Smirnov test. Intraclass correlation coefficient (ICC) performed with the SPSS statistical package (SPSS Interactive Graphics, v. 10.00, Chicago, Illinois) was used to assess intraobserver and interobserver variability; P < 0.05 was considered significant. RESULTS Neurological examination was normal in 15 patients. Five patients had slightly reduced Achilles tendon reflexes, and one patient had distal hypopallesthesia. Electroneurographic investigations of motor and sensory peripheral nerves (median, tibial, and sural bilaterally) showed normal findings, excluding involvement of the large myelinated peripheral fibers. Skin Biopsy. Compared to normal values (N), patient biopsies contained a significantly lower (P < 0.05) ENFD in the fingertip (3 6 1; N ¼ 13.1 6 410 Small Fiber Neuropathy in FD 8 mean value 6 SD), thigh (13.1 6 1; N ¼ 27 6 12), and leg (8 6 1; N ¼ 17.4 6 6). Epidermal nerve fibers showed morphological abnormalities such as axonal varicosities or swelling and nerve ending sprouts (Fig. 1E). There was a proximal-distal gradient of ENFD in patients. The distal site (i.e., leg) was significantly more affected (P < 0.001) than the proximal site (i.e., thigh), and older patients had a lower ENFD than did young patients (r ¼ 0.7; P < 0.05). Autonomic innervation was deficient in the dermal annexes. The results were equally severe in adrenergic nerves around AVAs (Fig. 1F), in MEP (Fig. 1G) and in cholinergic nerves around SG (Fig. 1H). The mean score of autonomic innervation in patients was reduced compared to normal (N) in the leg (1.8 6 0.7; N ¼ 3.7 6 0.4; P < 0.001), thigh (2.1 6 0.6; N ¼ 3.9 6 0.4; P < 0.001), and finger (2 6 0.4; N ¼ 3.9 6 0.4; P < 0.001) following a similar proximal-distal gradient (P < 0.001) as for ENFD. Autonomic and somatic innervations were similar in symptomatic and asymptomatic patients at each skin site (P > 0.5). The ICC for interobserver and intraobserver reproducibility of autonomic score analysis was 0.86 and 0.96, respectively (P < 0.0001), indicating a high level of reliability. DISCUSSION The clinical and pathological features of FD, including a length-dependent small fiber neuropathy, are well described in male patients.7 Recently, a high frequency of a multisystemic disease was reported in heterozygous females who carry the Fabry mutation and mainly complain of burning pain in the extremities.2,8 Direct evidence of small fiber neuropathy in female patients showed reduced ENFD.2 However, the type of neuropathy (i.e., length-dependent or ganglionopathy) was not defined due to lack of a proximal biopsy site (i.e., thigh). Autonomic dysfunction was excluded by indirect tests such as sympathetic skin response and heart rate variability, although patients often reported autonomic symptoms. Our study confirms the presence of small fiber neuropathy in FD females and adds new information to the previously reported peripheral nerve involvement. In particular, we showed that the small fiber neuropathy could be present before the onset of clinical symptoms, as has been reported in male patients9 in the absence of other organ dysfunction and comorbidities (i.e., renal failure, diabetes, etc.). The similar degree of skin innervation abnormalities between symptomatic and asymptomatic patients may suggest that the mechanisms underlying pain in FD patients is not a direct effect of skin denervation but is possibly MUSCLE & NERVE March 2010 C O L O R FIGURE 1. Skin innervation in a 31-year-old woman with FD and in an age-matched control. The control subject is represented in (A– D), whereas the female FD patient is shown in (E–H). (A,E) PGP-positive epidermal innervation: the patient (E) had reduced ENFD compared to the control (A), which was generally associated with morphological abnormalities of the epidermal innervation such as axonal varicosities (arrows), swelling (arrowheads), and nerve ending sprouts. (B,C,F,G) The DbH-positive adrenergic dermal innervation: the patient has a clear decrease of adrenergic nerve fibers of AVAs (F) and MEP (G) compared to the control (B,C, respectively). (D,H) VIP-positive cholinergic dermal innervation: a loss of cholinergic nerve fibers was found around SG in the patient (H), whereas the control (D) had abundant VIP-positive nerve fibers around sweat tubules. related to impaired cell membrane protein functions, including ion channel activity. The differences among patients could not be explained by a Small Fiber Neuropathy in FD specific gene mutation, since subjects who expressed the same gene mutation showed different clinical and skin innervation abnormalities. A MUSCLE & NERVE March 2010 411 further explanation could be given by the evaluation of the level of the enzyme activity but, unfortunately, we were not able to perform it. As described in males,7 female FD patients had a neuropathy with a proximal-distal gradient of more severe involvement of the leg in comparison with the thigh (a length-dependent neuropathy). Even so, the ENFD reduction was equally distributed in the leg (53%), thigh (51%), and fingertip (77%), suggesting diffuse pathological peripheral involvement. Our data indicated that the neuropathy in these female patients involved both somatic and autonomic peripheral nerve fibers. Our finding of autonomic involvement compared to a previous study2 could be explained by our direct evaluation of dermal annex innervation, which yielded a more detailed analysis of autonomic innervation. Our main conclusions are: (1) asymptomatic, heterozygous FD females with preserved renal function may have a small fiber neuropathy; and (2) small fiber neuropathy in female patients is a length-dependent neuropathy involving somatic and autonomic peripheral nerve fibers. We thank Prof. W.R. Kennedy for helpful comments and suggestions in revising the article, and Ms. A. Laffi helped with references and editing. We thank Ms. Anne Collins for the English revision of the article. R.L. and V.D. share responsibility for the integrity of the data and data analysis. Supported by RFO 2008 University of Bologna grant to R.L. REFERENCES 1. Mehta A, Ricci R, Widmer U, Dehout F, Garcia de Lorenzo A, Kampmann C, et al. Fabry disease defined: baseline clinical manifestations of 366 patients in the Fabry Outcome Survey. Eur J Clin Invest 2004; 34:236–242. 2. Laaksonen SM, Röyttä M, Jääskeläinen SK, Kantola I, Penttinen M, Falck B. Neuropathic symptoms and findings in women with Fabry disease. Clin Neurophysiol 2008;119:1365–1372. 3. Kennedy WR, Wendelschafer-Crabb G. The innervation of human epidermis. J Neurol Sci 1993;115:184–190. 4. Schütz B, Schäfer MK, Eiden LE, Weihe E. VIP and NPY expression during differentiation of cholinergic and noradrenergic sympathetic neurons. Ann N Y Acad Sci 1998;865:537–541. 5. Donadio V, Nolano M, Provitera V, Stancanelli A, Lullo F, Liguori R, et al. Skin sympathetic adrenergic innervation: an immunofluorescence confocal study. Ann Neurol 2006;59:376–381. 6. Donadio V, Nolano M, Elam M, Montagna P, Provitera V, Bugiardini E, et al. Anhidrosis in multiple system atrophy: a preganglionic sudomotor dysfunction? Mov Disord 2008;23:885–888. 7. Lacomis D, Roeske-Anderson L, Mathie L. Neuropathy and Fabry’s disease. Muscle Nerve 2005;31:102–107. 8. Moller AT, Feldt-Rasmussen U, Rasmussen AK, Sommer C, Hasholt L, Bach FW, et al. Small-fibre neuropathy in female Fabry patients: reduced allodynia and skin blood flow after topical capsaicin. J Peripher Nerv Syst 2006;11:119–125. 9. Morgan SH, Rudge P, Smith SJ, Bronstein AM, Kendall BE, Holly E, et al. The neurological complications of Anderson-Fabry disease (alpha galactosidase A deficiency)—investigation of symptomatic and presymptomatic patients. Q J Med 1990;75:491–504. A NOVEL CLCN1 MUTATION (G1652A) CAUSING A MILD PHENOTYPE OF THOMSEN DISEASE KISHORE R. KUMAR, MBBS,1 KARL NG, MBBS,1 HIMESHA VANDEBONA, BSc, PhD,2 MARK R. DAVIS, MHGSA, PhD,3 and CAROLYN M. SUE, MBBS, PhD1,2 1 Department of Neurology, Royal North Shore Hospital and University of Sydney, St. Leonards, New South Wales 2065, Australia 2 Neurogenetics, Kolling Institute, Royal North Shore Hospital and University of Sydney, New South Wales, Australia 3 Neurogenetics Unit, Department of Anatomical Pathology, Royal Perth Hospital, Perth, Western Australia, Australia Accepted 2 November 2009 ABSTRACT: We investigated a 62-year-old man who had mild clinical features of myotonia congenita. He was found to have a novel heterozygous G-to-A nucleotide substitution at position 1652 in exon 15 of the CLCN1 gene. Clinicogenetic studies performed on his family revealed that his asymptomatic son also shared the mutation. We conclude that a novel chloride channel mutation (G1652A) has caused a mild form of autosomal-dominant myotonia congenita (Thomsen disease) in this family. Muscle Nerve 41: 412–415, 2010 Myotonia congenita can be classified according to the mode of inheritance as either autosomal dominant (Thomsen disease) or recessive (Becker Abbreviations: APB, abductor pollicis brevis; bp, base pair(s); CLCN1, skeletal muscle voltage-gated chloride channel gene; CRD, complex repetitive discharge; CMAP, compound muscle action potential; CK, creatine kinase; EDX, electrodiagnostic; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism Key words: CLCN1; complex repetitive discharges; mild phenotype; myotonia; novel mutation; Thomsen myotonia congenita Correspondence to: C.M. Sue; e-mail: csue@med.usyd.edu.au C 2009 Wiley Periodicals, Inc. V Published online 30 October 2009 in Wiley InterScience (www. interscience.wiley.com). DOI 10.1002/mus.21610 412 A Novel CLCN1 Mutation disease). It is characterized by impaired muscle relaxation after forceful contraction (myotonia). This is secondary to a reduction in membrane chloride conductance that causes hyperexcitability of the skeletal muscle membrane and repetitive firing of muscle action potentials.1,2 Multiple mutations have been characterized in Thomsen disease patients and indicate a high degree of genetic heterogeneity.3 We report a 62-year-old man with mild clinical features of Thomsen disease who was found to have a novel mutation in the CLCN1 gene. We also assessed other members of the family and found one family member who shared the mutation. METHODS Family Studies. Clinicogenetic assessments were performed on six members of a single pedigree (see Fig. 1A). Neurophysiological studies were performed on the proband, his two children, and his sister. MUSCLE & NERVE March 2010 C O L O R FIGURE 1. (A) Pedigree of the family with Thomsen disease. Filled symbol: affected; open symbol: unaffected; open symbol with black square: asymptomatic carrier; arrow: proband; question mark: genetic status unknown. (B) Identification of heterozygous c.1652G>A point mutation in the CLCN1 gene in patient 2. The partial sequence electropherograms of the CLCN1 gene show: affected subject (proband’s son) (top); and wild-type sequence (bottom). (C) Assessment of c.1652G>A point mutation of the proband’s relatives by polyacrylamide gel electrophoresis. The figure shows an RFLP pattern of wild-type and mutant type when PCR product from CLCN1 gene was digested with Mbo1 restriction enzyme. Wild-type PCR product is digested into two bands [290 base pairs (bp) and 30 bp]. The c.1652G>A mutation creates a new restriction site resulting in four bands (290 bp, 174 bp, 116 bp, and 30 bp) in the heterozygous mutational status. The 30 bp band is not shown. M, marker; U, uncut PCR product (320 bp). (D) Protein alignment of a sequence encompassing the chloride channel 1 protein p.Gly551Asp (p.G551D) mutation, indicating conservation of the glycine residue among chloride channel 1 proteins from other species. Patient 1 (II-2) The patient is a 62-year-old man from an Australian family that had no recognized history of consanguinity or neuromuscular disorders. He had normal postnatal and early childhood development. His symptoms began at the age of 10 years when he noticed that he had trouble letting go after gripping a hand-rail tightly. He also had trouble releasing his hands from the oars after rowing. In the last 5–6 years he experienced occasional generalized stiffness and myalgia, worse in the mornings and improving with exercise. His symptoms were not aggravated by changes in temperature. On examination, he was noted to have mild atrophy of the shoulder girdle and the right calf. There was also some subtle orbicularis oculi weakA Novel CLCN1 Mutation ness, and there was evidence of percussion myotonia of the abductor pollicis brevis (APB) muscle. Magnetic resonance imaging confirmed atrophy of the posterior compartment of the right lower limb. His resting serum creatine kinase (CK) was normal. Patient 2 (III-3) Patient 1’s 37-year-old son had no symptoms of myotonia, but there was evidence of subtle weakness of orbicularis oculi and percussion myotonia of the APB. His serum CK was mildly elevated (371 U/L, normal <300). Other Family Members (II-1, III-1, III-2, and III4) These patients had no features of myotonia congenita on clinical assessment. In each of these family members, the serum CK was normal. DNA was extracted from the patients’ blood. We performed polymerase chain Mutational Analysis. MUSCLE & NERVE March 2010 413 reaction (PCR) and direct nucleotide sequencing for mutations in the CLCN1 gene. In the proband, the entire coding region of the CLCN1 gene was sequenced. In the proband’s relatives, exons 3, 4, 5, 7–13, 15, 17, and 23 were sequenced. Restriction fragment length polymorphism (RFLP) analysis was performed on PCR product from the CLCN1 gene in the proband’s relatives. Studies. An electrodiagnostic (EDX) assessment was performed on the proband, his two children, and his sister. Family members III-1 and III-2 declined to participate in the EDX studies. The EDX studies included standard nerve conduction studies and electromyography (sampling the right deltoid, iliopsoas, vastus medialis, tibialis anterior, medial head of gastrocnemius and left thoracic paraspinal muscles). Electromyography of the right APB was performed before and after cooling to <20 C using an ice pack. Repetitive stimulation at rates of 2 and 10 HZ was applied to the right ulnar nerve, with 10 stimuli delivered in each train. Exercise testing was performed sequentially on the right and left abductor digiti minimi. The long exercise test was performed as described by McManis and colleagues.4,5 A short exercise test (single and repeated trials) was performed as described by Streib and colleagues.4,6 Five pre-exercise compound muscle action potentials (CMAPs) were recorded, and a repeat short exercise test was performed after cooling to <20 C using an ice pack and after rewarming to 32 C using a heat pack. Neurophysiological RESULTS Direct sequencing identified a novel heterozygous G-to-A nucleotide substitution at position 1652 in exon 15 at codon 551 of the CLCN1 gene in the proband and his son (see Fig. 1B). The mutation was confirmed on RFLP analysis (see Fig. 1C). This mutation changed a highly conserved region of the chloride channel gene (see Fig. 1D). No additional mutations were identified, and the mutation was not found in other family members. Mutation Analysis. EDX Studies. Electromyography revealed complex repetitive discharges (CRDs) without any true myotonic discharges in several muscles of both the proband and his son. Electromyography was normal in the proband’s daughter and sister. Cooling of APB did not change the electromyographic findings in any of the family members studied. The other neurophysiological tests were normal in all family members with a <10% decrement on repetitive nerve stimulation, a decrease in CMAP amplitude from peak of <40% for the long exercise test,5 and a maximum change in the CMAP amplitude between 10% and þ20% of the pre-exercise 414 A Novel CLCN1 Mutation value for the short exercise test.4 The CMAP amplitude did not change with repeated trials, and there was a normal response to cooling7 and rewarming. DISCUSSION In this study we have reported a novel chloride channel mutation that causes autosomal-dominant myotonia congenita. It is unlikely that the mutation is a benign polymorphism given that it occurred in a highly conserved region of the chloride channel gene and caused a significant change in amino acid chemistry. The majority of described amino acid changes in this gene are pathogenic. The family studies are also consistent with a pathogenic mutation. The pathogenicity of the mutation could be further explored with electrophysiological expression studies of chloride channel function.3 There were some distinct clinical features in the proband. He had a mild clinical phenotype with a late onset of symptoms (compared with the mean age of onset of 6 years3) and few symptoms and signs in adulthood. Also, Thomsen disease is usually characterized by muscle hypertrophy rather than atrophy, although a ‘‘dystrophic’’ variant of myotonia congenita with distal muscle atrophy has been described.8 CRDs are thought to arise from ephaptic firing of a small group of muscle fibers through a reentrant mechanism, with one fiber acting as a principal pacemaker.9,10 CRDs do not have the waxingand-waning pattern of myotonic discharges, although it can be difficult to differentiate CRDs from myotonic discharges given that they share a similar discharge frequency. Although CRDs are known to be secondary to some myopathic conditions,9 to our knowledge they have not previously been associated with myotonia congenita. The CRDs in this report probably represent a forme fruste of true myotonic discharges. More characteristic electrophysiological findings in myotonia congenita include an abnormal short exercise test with a mild increase in CMAP amplitude during exercise and a reduction of the CMAP amplitude following exercise,5,11,12 a CMAP decrement with repetitive nerve stimulation (delivered at 10 HZ or more),11 and widespread myotonic discharges at rest and during volitional activity.11 The proband’s son was asymptomatic and probably represents a subclinical variant of Thomsen disease. Heterozygotes for the chloride channel gene (CLCN1) mutation can have a broad phenotypic spectrum, even between heterozygous members of the same family. They can be asymptomatic with mild myotonia revealed only by physical examination.13 There may also be a gene dosage effect; MUSCLE & NERVE March 2010 if two of these genes were inherited, a more severe phenotype would most likely emerge.14 REFERENCES 1. Sasaki R, Ito N, Shimamura M, Murakami T, Kuzuhara S, Uchino M, et al. A novel CLCN1 mutation: P480T in a Japanese family with Thomsen’s myotonia congenita. Muscle Nerve 2001;24:357–363. 2. Fahlke C, Beck CL, George AL. A mutation in autosomal dominant myotonia congenita affects pore properties of the muscle chloride channel. Proc Natl Acad Sci USA 1997;94:2729–2734. 3. Fialho D, Schorge S, Pucovska U, Davies NP, Labrum R, Haworth A, et al. Chloride channel myotonia: exon 8 hot-spot for dominant-negative interactions. Brain 2007;130:3265–3274. 4. Fournier E, Arzel M, Sternberg D, Vicart S, Laforet P, Eymard B, et al. Electromyography guides toward subgroups of mutations in muscle channelopathies. Ann Neurol 2004;56:650–661. 5. McManis PG, Lambert EH, Daube JR. The exercise test in periodic paralysis. Muscle Nerve 1986;9:704–710. 6. Streib EW, Sun SF, Yarkowski T. Transient paresis in myotonic syndromes: a simplified electrophysiologic approach. Muscle Nerve 1982;5:719–723. A Novel CLCN1 Mutation 7. Kuntzer T, Michel P. Muscle membrane polarization after provocative tests, and after cooling: the normal CMAP changes to be expected. Clin Neurophysiol 2004;115:1457–1463. 8. Nagamitsu S, Matsuura T, Khajavi M, Armstrong R, Gooch C, Harati Y, et al. A ‘‘dystrophic’’ variant of autosomal recessive myotonia congenita caused by novel mutations in the CLCN1 gene. Neurology 2000;55:1697–1703. 9. Fellows LK, Foster BJ, Chalk CH. Clinical significance of complex repetitive discharges: a case–control study. Muscle Nerve 2003;28: 504–507. 10. Trontelj J, Stålberg E. Bizarre repetitive discharges recorded with single fibre EMG. J Neurol Neurosurg Psychiatry 1983;46:310–316. 11. Amato AA, Dumitru D. Hereditary myopathies. In: Dumitru D, Amato AA, Zwarts M, editors. Electrodiagnostic medicine, 2nd ed. Philadelphia: Hanley & Belfus; 2002. p 1301. 12. Kuntzer T, Flocard F, Vial C. Exercise test in muscle channelopathies and other muscle disorders. Muscle Nerve 2000;23:1089–1094. 13. Colding-Jorgensen E. Phenotypic variability in myotonia congenita. Muscle Nerve 2005;32:19–34. 14. Bernard G, Poulin C, Puymirat J, Sternberg D, Shevell M. Dosage effect of a dominant CLCN1 mutation: a novel syndrome. J Child Neurol 2008;23:163–166. MUSCLE & NERVE March 2010 415