Myotonia congenita: Novel mutations in CLCN1 gene and functional characterizations in Italian patients

Journal of the Neurological Sciences 318 (2012) 65–71

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Myotonia congenita: Novel mutations in CLCN1 gene and functional characterizations in Italian patients Gianna Ulzi a, 1, Marzia Lecchi b, 1, Valeria Sansone c, Elisa Redaelli b, Eleonora Corti b, Domenica Saccomanno a, Serena Pagliarani a, Stefania Corti a, Francesca Magri a, Monika Raimondi d, Grazia D'Angelo e, Anna Modoni f, Nereo Bresolin a, e, Giovanni Meola c, Enzo Wanke b, Giacomo P. Comi a, Sabrina Lucchiari a,⁎ a

Dino Ferrari Centre, Department of Neurological Sciences, University of Milan, IRCCS Fondazione Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy c IRCCS Policlinico San Donato, University of Milan, S. Donato Milanese, Milan, Italy d Ospedale Regionale di Lugano, Centro di Medicina del Sonno, Lugano, Switzerland e Scientific Institute IRCCS Eugenio Medea, Bosisio Parini, Lecco, Italy f Department of Neuroscience, Catholic University, Rome, Italy b

a r t i c l e

i n f o

Article history: Received 17 February 2012 Received in revised form 27 March 2012 Accepted 29 March 2012 Available online 21 April 2012 Keywords: Skeletal muscle Channelopathy CLCN1 gene Myotonia congenita Thomsen's disease Becker's generalized myotonia

a b s t r a c t Myotonia congenita is an autosomal dominantly or recessively inherited muscle disorder causing impaired muscle relaxation and variable degrees of permanent muscle weakness, abnormal currents linked to the chloride channel gene (CLCN1) encoding the chloride channel on skeletal muscle membrane. We describe 12 novel mutations: c.1606G>C (p.Val536Leu), c.2533G>A (p.Gly845Ser), c.2434C>T (p.Gln812X), c.1499T>G (p.E500X), c.1012C>T (p.Arg338X), c.2403+1G>A, c.2840T>A (p.Val947Glu), c.1598C>T (p.Thr533Ile), c.1110delC, c.590T>A (p.Ile197Arg), c.2276insA Fs800X, c.490T>C (p.Trp164Arg) in 22 unrelated Italian patients. To further understand the functional outcome of selected missense mutations (p.Trp164Arg, p.Ile197Arg and p.Gly845Ser, and the previously reported p.Gly190Ser) we characterized the biophysical properties of mutant ion channels in tsA cell model. In the physiological range of muscle membrane potential, all the tested mutations, except p.Gly845Ser, reduced the open probability, increased the fast and slow components of deactivation and affected pore properties. This suggests a decrease in macroscopic chloride currents impairing membrane potential repolarization and causing hyperexcitability in muscle membranes. Detailed clinical features are given of the 8 patients characterized by cell electrophysiology. These data expand the spectrum of CLCN1 mutations and may contribute to genotype–phenotype correlations. Furthermore, we provide insights into the fine protein structure of ClC-1 and its physiological role in the maintenance of membrane resting potential. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Myotonia congenita (MC) belongs to the group of non-dystrophic myotonia and can be inherited either by an autosomal dominant (Thomsen's disease OMIM 160800) or recessive manner (Becker's disease, OMIM 255700). It is characterized by impaired muscle relaxation after voluntary contraction and variable degrees of muscle weakness. It is caused by mutations in the CLCN1 gene (RefSeq NC_000007.13) on chromosome 7q35 encoding the major skeletal muscle chloride channel CLC-1 (RefSeq NM_000074.2). It is well established that chloride channels play a role in the regulation of the muscle membrane and thus

⁎ Corresponding author at: “Dino Ferrari Centre”, Department of Neurological Sciences, University of Milan, Fondazione I.R.C.C.S. Ca´ Granda Ospedale Maggiore Policlinico, Via Sforza 35, 20122 Milano, Italy. Tel.: +39 02 55033843; fax: +39 02 503020430. E-mail address: [email protected] (S. Lucchiari). 1 These authors contributed equally to this work. 0022-510X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2012.03.024

participate in the maintenance of the resting potential. Their inactivation by mutations modifies the cycle of excitability of the muscle membrane, shifting it towards hyperexcitability by slowing the return of the membrane to the resting potential after depolarization. Myotonia is directly correlated to the repetitive activation of sodium channels caused by this state of hyperexcitability [1–3]. Each muscle chloride channel comprises two identical protein molecules, each constituting a separate ion conductance pathway, the socalled protopore. In autosomal recessive myotonia congenita, both subunits have a disease-causing mutation. This results in chloride channel reduction to 40% or less, which is sufficient to cause myotonic contractions. Autosomal dominant myotonia congenita is believed to result from the presence of one dominant-negative mutation that modifies either the gating of both protopores or the selectivity of one of the two protopores [4–6]. However, some mutations have been found to lead to autosomal dominant myotonia congenita in some patients, and to a homozygous recessive form in others.

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On clinical grounds, the dominant and recessive forms may be indistinguishable [7]. In vivo electrophysiological pattern responses to exercise test protocols have been described to cluster patients with different channelopathies, including chloride channelopathies [8], but electromyography does not distinguish between the recessive and the dominant phenotype. A recent clinical and genetic study of MC [9] highlighted the clinical similarities between dominant and recessive forms and suggested the existence of a possible hot-spot mutation region on exon 8 indicating that it has an important role in dominant-negative interactions between the two monomers constituting the chloride channel. This study also confirmed the genetic heterogeneity of this condition, and suggests that the greater the number of pathogenic mutations described the more accurate will be the genetic counseling. We first report on 12 novel mutations: c.1606G>C (p.Val536Leu), c.2533G>A (p.Gly845Ser), c.2434C>T (p.Gln812X), c.1499T>G (p.E500X), c.1012C>T (p.Arg338X), c.2403+1G>A, c.2840T>A (p.Val947Glu), c.1598C>T (p.Thr533Ile), c.1110delC, c.590T>A (p.Ile197Arg), c.2276insA Fs800X, c.490T>C (p.Trp164Arg) on CLCN1 gene in 22 unrelated Italian patients. Furthermore the following mutations found in eight patients were considered for functional analysis: p.Trp164Arg, p.Ile197Arg and p.Gly845Ser, together with the previously reported p.Gly190Ser [10] which was present in 5 patients from our cohort. All the tested mutations, with the exception of p.Gly845Ser, caused alterations in the functional characteristics of hClC-1 channels, which consisted in modifications in the voltage-dependence of open probability, in the deactivating behavior and in pore properties. These defects in the physiology of hClC-1 channels were discussed in relation to the molecular background and the clinical features observed in myotonic patients.

2.2. Mutational screening Genomic DNA was extracted from peripheral leukocytes using FlexiGene DNA Kit (QIAGEN, Hamburg, Germany, www.qiagen.com) following manufacturer's instruction. The 23 coding exons of CLCN1 gene and the respective intron-junctions were PCR-amplified with specific primer pairs. Amplification products were sequenced by Big Dye Terminator Cycle Sequencing Kit (Applied Biosystem, Foster City, USA, www.appliedbiosystems.com) in an automated sequencer 3100 (Applied Biosystem). The sequences were compared to GenBank sequence NM_000083 using SeqScape software (Applied Biosystem). To better resolve the signals yielded by direct sequencing amplification products of exon 5 from patient 4 were cloned in TOPO-TA Cloning System (Invitrogen, Carlsbad, CA, USA, www.invitrogen.com). For any mutation found, two independent amplification and sequencing rounds were performed. For each candidate missense mutation identified 200 Italian control chromosomes were screened and evolutionary comparison was performed (Fig. 1). 2.3. Mutagenesis

2. Materials and methods

The wild type full-length cDNA of CLCN1 was amplified by Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany) and the forward primer 5′-ATGGAGCAATCCGGTCACAG-3′ and the reverse primer 5′-TCAAAGGATCAGTTCATCCTCAT-3′. The p.Trp164Arg, p.Gly190Ser, p.Ile197Arg and p.Gly845Ser point mutations were introduced into human CLC-1 cDNA to be cloned in pcDNA3.1/V5His TOPO TA Expression Kit (Invitrogen) vector by using Quickchange TM Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). Two independent mutant clones were tested for each mutation. Sequences of the four primer pairs for mutagenesis are listed in Table 1. Wild type and mutated cDNAs were expressed in tsA cells for functional characterization.

2.1. Patients

2.4. Cell electrophysiology

Twenty-two unrelated patients (15 males, 7 females, mean age at examination: 35 years; age range: 7–68) fitting the criteria for either Thomsen or Becker disease [1,11] were included in the present study. Written informed consent was designed according to the Declaration of Helsinki and obtained from all patients and healthy subjects participating in the study. Genomic DNA was obtained from patients and their family members, when available. Trinucleotide repeat expansions in the DMPK gene associated with myotonic dystrophy type 1 and tetranucleotide repeat expansions in intron 1 of the ZNF9 gene associated with myotonic dystrophy type 2 were ruled out in all patients.

TsA cells, a subclone of human embryonic kidney cells, were cultured in DMEM/F-12 (Ham) (Sigma) supplemented with 10% fetal calf serum and maintained at 37 °C with 5% CO2. For the experiments, cells were plated in 35-mm culture dishes and transfected by Lipofectamine (Invitrogen). About 0.3 μg of the plasmid pcDNA3.1/ V5-His-TOPO was used for each dish and was cotransfected with 0.1 μg of green fluorescent protein plasmid E-GFPpcDNA3 (Clonotech Laboratories, USA). Current recordings were performed 48–72 h after the transfection by the patch-clamp technique in the whole-cell configuration, using the MultiClamp 700A amplifier and pClamp 8.2 software (Axon Instruments, USA) for data acquisition. Agar bridges

Fig. 1. Comparative analysis of mutated amino acids selected for this study and surrounding sequences indicating a high degree of conservation.

G. Ulzi et al. / Journal of the Neurological Sciences 318 (2012) 65–71

were used to connect the bath solution to the amplifier in order to avoid junction potentials. Pipette resistance was about 1.3–1.8 MΩ; capacitance and series resistance errors were compensated (85– 90%) before each protocol run to reduce voltage errors to less than 5% of the protocol pulse. The standard pipette solution contained (mM): cesium aspartate 128, MgCl2 4, EGTA 10, HEPES 10 at pH7.30. The external solution was prepared with (mM): NaCl 164, CaCl2 2, MgCl2 1, HEPES 5 at pH 7.40. The voltage-dependence of the relative open probability (Po) was obtained according to the protocol proposed in [12] and modified as described in [13]. Briefly, after 1.5-s prepulses from −140 mV to 100 mV, tail currents were generated at −135 mV. The peaks of tail currents at the preconditioning potentials (Iv) were normalized at the maximal current (Imax), to obtain the relative Po. Data were successively plotted as a function of preconditioning voltages and fitted by a Boltzmann function:     Iv ¼ Io þ ðImax Io Þ= 1 þ exp V1=2  V =k ;

where Io is a constant offset, V1/2 is the half-maximal activation voltage and k is the slope factor. Deactivation channel properties were determined as reported in [14], holding the cells at +40 mV for 100 ms and then changing the voltage test from +80 mV to −160 mV. The time courses of current deactivation were determined by fitting current traces with a twoexponential function: IðtÞ ¼ A1 expð−t=τ1 Þ þ A2 expð−t=τ2 Þ þ C; where τ1 and A1 are the time constant and the amplitude of the slow component and τ2 and A2 are the same parameters but for the fast component; C is a constant. The fractional amplitudes of the deactivating current components were calculated by dividing the component amplitude by the peak current amplitude (Imax = A1 + A2 + C), as previously explained [15]. The instantaneous current–voltage relationship was obtained by the extrapolation of fitted current traces to the beginning of each test voltage step. The isochronal current–voltage relationship was determined by current measurements at the end of each test voltage step. All data are presented as mean ± SEM. Statistical evaluation was performed using the one-way and the two-way analysis of variance (ANOVA) with the post hoc Tukey test. Statistical significance was set at p b 0.05. 3. Results Detailed clinical examination and genetic outcomes (Table 2) were described only for the patients (1–8) whose mutations have been subjected to functional analysis.

Table 1 Sequences of the primers used for mutagenesis. Mutation

Sequence

W164R-forward W164R-reverse G190S-forward G190S-reverse I197R-forward I197R-reverse G845S-forward G845S-reverse

5′-TTCCTCTGCAGTTCCTGGTCCGGGTCACCTTCCCACTAGTC-3′ 5′-GACTAGTGGGAAGGTGACCCGGACCAGGAACTGCAGAGGAA-3′ 5′-CTCCCCAGGCTGTTGGCTCTTCAATCCCCGAAATGAAGACAA-3′ 5′-TTGTCTTCATTTCGGGGATTGAAGAGCCAACAGCCTGGGGAG-3′ 5′-AATCCCCGAAATGAAGACAAGACTTCGTGGGGTTGTCCTGA-3′ 5′-TCAGGACAACCCCACGAAGTCTTGTCTTCATTTCGGGGATT-3′ 5′-ATACCCTGTTTTCACTCCTTAGCCTCCACCTCGCTTACGTG-3′ 5′-CACGTAAGCGAGGTGGAGGCTAAGGAGTGAAAACAGGGTAT-3′

Listed in the table are the primers used for mutagenesis experiments on CLCN1 cDNA inserted in pcDNA3.1/V5-His TOPO TA vector.

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3.1. General clinical features Age at onset was mainly in the first and second decades. The predominant symptom at onset was generalized stiffness and in half of the patients this was mostly localized in the lower limbs. Neither grip myotonia nor myotonia affecting the facial and jaw muscles or the tongue were major complaints in these patients. Warm-up was present in most cases and triggers for myotonia were in general rest and low outside temperature. Muscle pain was a common feature in about half of the patients. This was described as myalgias in the lower limbs rather than as cramps and was usually independently described from myotonia. The time-lag between age at onset of symptoms and age at examination was long, ranging from 10 to 30 years, in general because the patients reported that myotonia or muscle weakness, when present, was not severe enough to limit them functionally and thus never caused them to seek medical attention. The higher proportion of affected males in our study population (3:1 ratio) is in agreement with literature data, indicating that skeletal chloride channelopathies affect males more than females [16,17]. Permanent muscle weakness affecting lower limbs was present in one-third of patients while transient episodes were present in 25% of patients and were triggered in all cases by exercise. 3.2. Genetics Sequencing analysis detected twelve novel mutations localized in the schematic protein in Fig. 2, modified from Ma [18]. Genetic screening was carried out for both parents of each patient whose cell electrophysiology has been performed, with the exception of patients 7 and 8. Patient 7 concern mutation screening could only find Ile197Arg, but we cannot exclude macro-rearrangements. Indeed in the second part of CLCN1 gene, neither homozygous nor heterozygous polymorphisms indicating the presence of both paternal and maternal intact gene sequences were detected. Given that exerciseinduced myotonia, absence of warm-up phenomenon and presence of muscle pain could be also suggestive of sodium channel disorder, screening of SCN4A gene was performed, and resulted negatively. Parents of patient 8 were not living anymore. The nucleotide variations on the second allele in patients 1, 3, 4 and 5 are respectively one skipping and three nonsense mutations, which can be considered pathogenic regardless of the cells' electrophysiological behavior. 3.3. Cell electrophysiology Among the new mutations described in this paper, Trp164Arg, Gly190Ser, Ile197Arg and Gly845Ser presented a common feature: the substitution of a non-polar amino acid with a polar residue. In order to investigate the functional consequences of these substitutions, we expressed the cloned cDNAs in tsA cells. All the mutants were able to evoke currents in the range of potentials from −140 to 100 mV (Fig. 3A). Tail current densities, elicited at −135 mV, resulted in: 266 ± 31 pA/pF for Trp164Arg (n = 11), 214 ± 26 pA/pF for Gly190Ser (n = 14), 391 ± 105 pA/pF for Ile197Arg (n = 12) and 329 ± 58 pA/pF for Gly845Ser (n = 14) (Table 3). Only for Gly190Ser we observed a significant reduction (pb 0.05) compared to WT ClC-1 (351± 57 pA/pF, n = 13). The open probability of the functional channels (Po) was studied by applying the protocol described in 2.4. Representative current traces and the corresponding Po-voltage relationship are shown in Fig. 3. Mutant channels Trp164Arg, Gly190Ser and Ile197Arg opened to a lesser extent, exhibiting a smaller relative Po at membrane voltages within the physiological range (Fig. 3B). Compared to WT channels, the voltage-dependence of their open probability was significantly shifted to the right, respectively by 14.9 mV for Trp164Arg, 28.2 mV for Ile197Arg and 29 mV for Gly190Ser (p b 0.05). The V1/2

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Table 2 Detailed clinical features of subjects with mutations characterized by cell electrophysiology. 1

2

3

4

5

6

7

8

Sex Age at examination Symptom at onset Age at onset (decade) Clinical myotonia Lids Tongue Jaw muscles Neck muscles Hands Legs Warm–up Lid–lag Paradoxical myotonia Triggers Bone contractures Muscle mass Hypertrophy

M 36 >LL stiffness 1st ++ + + – ++ +++ ++ + – Change in temperature + + Generalized – – – + Change in temperature Rest – MEX ++ x2 – ++Diffuse Type II fiber deficiency

F 38 >LL stiffness 1st ++ ++ – – ++ – + – – Rest cold – ++ LL + ++ + – – – Hyper thyroidism None None x2 – ++ Type IIB fiber deficiency

F 29 >LL stiffness 1st – – – – ++ ++ + – – – – +

M 18 Lid myotonia 1st + + – – ++ +++ ++ + – Rest – –

F 29 Hand myotonia 1st – + – – ++ – ++ – – – – –

M 18 > LL stiffness 1st + – – – ++ – ++ – – – – –

M 36 Muscle pain 2nd – – – – – ++ – – – Exercise – ++LL

F 68 >LL stiffness 3rd – – – – + + + + – Rest cold – –

– – – – – – – None None Normal – + nd

– – + – – – – None None x2 – ++ Type 1 predominance, type1 and 2 hypotrophy

– – – – – – – None None Normal – ++ nd

– ++ – + Exercise Rest Frontal balding None None x2 nd nd Fiber type diameter variability, increased central nuclei

– +++ – – – – Depression MEX ++ Normal – ++ nd

c.1471 + 1 G>A (intron 13) p.Trp164Arg (exon 4)

p.Gly190Ser (exon 5) nd

+ – – – Rest Exercise BBB, 80% FVC MEX ++ x2 – ++ Fiber type diameter variability, type 1 predominance, type 2 atrophy p.Gly190Ser (exon 5) p.Arg338X (exon 9)

p.Gly190Ser (exon 5) 1183_1187del fs427X (exon 11)

p.Gly190Ser (exon 5) p.Phe167Leu (exon 4)

p.Ile197Arg (exon 5) nd

p.Phe167Leu (exon 4) p.Gly845Ser (exon 22)

Hypotrophy Muscle pain Muscle weakness

Permanent Transient Triggers Alleviating factors

Other clinical findings Treatment for at examination myotonia Response to Treatment CK Electrophysiology Myopathic changes studies Myotonic discharges Muscle biopsy

Mutations

p.Gly190Ser (exon 5) p.E500X (exon 14)

Clinical, genetic and laboratory data of 8 Italian patients with MC whose mutations underwent cell electrophysiological analysis. M = male; F = female; > = predominant; LL = lower limb; ‘-’ = absent; ‘+’ = mild; ‘++’ = moderate; ‘+++’ = severe; na = not available; nd = not done; bold = new mutations reported here; MEX = mexiletine; FVC = forced vital capacity; x2 = two-fold normal values.

G. Ulzi et al. / Journal of the Neurological Sciences 318 (2012) 65–71

Patient ID

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Fig. 2. Cartoon of the skeletal muscle chloride channel and the 12 novel mutations found in our cohort of patients.

for the activation were respectively: − 42.6 ± 4.5 mV for Trp164Arg (n = 11), −29.3 ± 1.9 mV for Ile197Arg (n = 12), − 28.5 ± 1.8 mV for Gly190Ser (n = 14) and − 57.5 ± 0.7 mV for WT (n = 13) (Table 3). The correspondent slope factors were: 41.1± 4.2 mV for Trp164Arg, 43.5 ± 1.8 mV for Ile197Arg, 43.6 ±1.7 mV for Gly190Ser and 26.9 ± 0.6 mV for WT channels (Table 3). The same three mutations also influenced the kinetics of hClC-1 channels. The protocol used to study deactivation properties identified a different behavior compared to WT channels and showed relaxing currents starting from 0 mV (Fig. 4A). By fitting the current traces with a double-exponential curve, it was possible to dissect the characteristics

of the fast and the slow deactivation components. Deactivation time constants for WT ClC-1 followed the same trend previously described by [15] and are not shown in this paper. As concerns the mutants, while the slow deactivating time constant (τ1) remained unaltered compared to WT channels, the fast deactivating time constant (τ2) was significantly reduced in the potential range from −100 to −160 mV for Trp164Arg and Gly190Ser and from −60 to −160 mV for Ile197Arg (p b 0.05). Fractional current amplitudes display altered voltage dependence and significantly higher relative amplitudes for the slow (A1) and fast (A2) deactivating components were observed for these mutant channels between −80 and 0 mV (pb 0.05) (Fig. 5A). Significant reductions of the nondeactivating component C were also evident in the range of potentials from −80 and −40 mV (pb 0.05) (Fig. 5B). These data indicated that, at physiological membrane voltages, a wider percentage of channels underwent fast and slow deactivation when one of the substitutions Trp164Arg, Ile197Arg or Gly190Ser was present. The analysis of the isochronal I-V relationships revealed major variations in the depolarized and very hyperpolarized potentials but only minor variations (in the case of Trp164Arg and Gly190Ser) or no variations (for Ile197Arg) at physiological potential values (Fig. 4B). The instantaneous I-V relationship for all of the three mutants displayed, in contrast to the pronounced inward rectification of WT ClC-1, inward rectification at negative voltages and outward rectification in the positive potential range (Fig. 4B). The shift in the reversal membrane potential (Erev), described in Table 3, also suggested a loss in ionic selectivity [19]. Despite the position of the substitution, Gly845Ser mutant channels did not show any significant difference with WT ClC-1. In fact they deactivate upon hyperpolarization and activate further upon depolarization, the profiles of their currents superimposed with WT currents (Figs. 3A and 4A) and no changes in the voltage-dependence of opening was observed (V1/2 for activation, −54.6 ± 2.5 mV; slope factor, 24.1 ± 1.2 mV, n = 11, Table 3). The deactivation mechanisms and the IV relationships were also not modified compared to WT channels (Figs. 4B and 5). 4. Discussion

Fig. 3. Activation properties of mutant Trp164Arg, Gly190Ser, Ile197Arg and Gly845Ser hClC-1 channels. A) Representative currents evoked from mutant channels are shown and compared to WT current traces. Dash lines indicate the level of zero current. B) The corresponding Popen curves, obtained from the protocol and the traces in the panel A, are represented.

We have identified 12 novel mutations out of 22 individuals, confirming the high degree of clinical heterogeneity in patients with myotonia congenita. The clinical presentation of the eight patients

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Table 3 Electrophysiological properties of WT hClC-1 and mutants expressed in tsA cells. ClC-1 channels

V1/2, mV

Slope factor, mV

Erev from isochronal IV, mV

Erev from instantaneous IV, mV

Current density pA/pF

No. of cells

WT W164R G190S I197R G845S

− 57.5 ± 0.7 − 42.6 ± 4.5* − 28.5 ± 1.8* − 29.3 ± 1.9* − 54.6 ± 2.5

26.9 ± 0.6 41.1 ± 4.2* 43.6 ± 1.7 * 43.5 ± 1.8* 24.1 ± 1.2

− 34.8 ± 2.8 − 33.1 ± 4.3 − 41.3 ± 2.8 − 38.1 ± 3.1 − 31.0 ± 1.1

− 21.6 ± 2.0 − 5.8 ± 2.8** − 7.6 ± 3.1** 7.8 ± 3.2** − 17.2 ± 3.4

351 ± 57 266 ± 31 214 ± 26* 391 ± 105 329 ± 58

13 11 14 12 11

All values are means ± SEM. **Significantly different from WT with p b 0.01 *Significantly different from WT with p b 0.05.

described in detail in the present report does not differ from the one described in previous reports in patients with MC. Myotonia was mostly generalized, predominantly affecting the lower limbs and only very mildly affecting the facial, jaw and tongue muscles. Other common clinical findings were muscles of increased bulk and variable degrees of permanent muscle weakness. The 164 tryptophan has a high degree of conservation, as shown in Fig. 2. The novel mutation in patient 1 is located in a region containing a stretch of amino acids showing a high degree of conservation. This is confirmed by mutation p.Phe167Leu, which we found in patients 6 and 8, and p.Val165Gly both causing small shift of potential as reported by the electrophysiological study from Zhang [13]. Functional analysis of the resulting channels revealed alterations in their principal biophysical properties. They showed reduced open probability; at +40 mV open channels were less than 90% and even more depolarized potentials could not succeed in a complete activation. Half activation was shifted of about 15 mV towards positive potential values. Deactivation kinetics was also altered in the physiological range of muscle membrane potentials. The fractional amplitude of the fast and slow deactivating components showed, with the potential, a linear trend and higher values from −80 and 0 mV; moreover, in the physiological range of potentials, the nondeactivating component C was significantly reduced. This behavior indicated that mutant channels had a higher probability to relax with both fast and slow mechanisms. These modifications in the channel gating would reduce the availability

of chloride channels able to control muscle cell excitability and the consequence would be particularly relevant as all the effects we observed occurred in the physiological range of membrane potentials. The phenotype severity is justified both by the functional changes described and the presence on the second allele of an inframe skipping mutation of exon 13 lying in domain N. Actually, the disease onset was in infancy, and the patient has developed diffused myotonia (GM) as the Becker patient described by Meyer-Kleine [20] carrying the same mutation. Remarkably the missense p.Gly190Ser was detected in 5 out of 22 patients in our Italian cohort (23%). This mutation was first described in a large consanguineous Arab family [10] in both heterozygous and homozygous forms. It is sited on exon 5, which falls in the D-domain and it implies a neutral side chain to turn into a polar one. It might be of interest that this amino acid change is due to a double nucleotide substitution, c.568_569 GG>TC, which lies in the consensus sequences where the spliceosome complex binds, and thus may affect the splicing efficiency. Moreover, Gly190 is located in a well conserved motif, the GSGIPEMK sequence, which is considered of particular importance for two channel properties: anion selectivity and single channel conductance [19,21]. Indeed our experiments showed that mutant Gly190Ser affected open probability, deactivation properties, permeability and reduced current densities. The last effect could be related to alteration in splicing efficiency turning in a reduced membrane insertion efficiency. Unfortunately, RNA was not available for further study splicing.

Fig. 4. Pore properties of mutant Trp164Arg, Gly190Ser, Ile197Arg and Gly845Ser hClC-1 channels. A) Representative currents evoked from WT and mutant channels are shown with the deactivating protocol (see 2.4 for details). Zero current levels are indicated as dash lines. B) Isochronal I-V and instantaneous I-V relationships for the WT and the mutants. The corresponding reversal membrane potentials (Erev) are described in Table 3.

Fig. 5. Deactivation properties of mutant Trp164Arg, Gly190Ser, Ile197Arg and Gly845Ser hClC-1 channels are represented. A) Voltage-dependencies of the slow (A1) and fast (A2) deactivating components. B) Nondeactivating component C.

G. Ulzi et al. / Journal of the Neurological Sciences 318 (2012) 65–71

The phenotypes observed in patients 4 and 2 were similar in age at onset, distribution, temperature-sensitivity and severity of myotonia. Patient 4, also carried the nonsense p.Arg338X. At this position an amino acid substitution was previously reported (p.Arg338Gln) [22] resulting in a quite severe phenotype with dysphagia and hypertrophy of the lower limbs. p. Gly190Ser is likely to be a recessive mutation, and we can speculate that a mutation in the second allele was probably missed in patient 2. Patients 3, 5, and 6 showed a recessive pattern of inheritance and shared most of the clinical signs (Table 2), being heterozygous compounds of Gly190Ser and, respectively, Glu500X, fs427X, and Phe167Leu. The first two variations are severe and they are predicted to give no protein products while functional expression of p.Phe167Leu had shown a small shift of the voltage dependence of the ClC-1 open probability [13]. Mutation p.Ile197Arg described in patient 7 is a novel mutation sited in domain D and quite conserved during evolution as well the other missense herein described (see Fig. 1). As previously described for Trp164Arg, the substitution in residue 197 caused changes in ClC1 functional properties which are likely to reduce macroscopic chloride currents as it generally demonstrated for MC related mutants. As mentioned in Section 3.2, a second mutation could not be found, nonetheless the presence of transient weakness and marked lower limb hypertrophy is in favor of a recessive condition, though a dominant inheritance can not be excluded. Indeed, it should be considered that p.L198V was previously reported [23] as possibly dominant. The p.L198V is near the helix belonging to the dimer interface where most mutations were found to effect the common gate [24,25]. Unfortunately a family study could not be performed to define the actual inheritance pattern of our mutation. Patient 8 carried the novel p.Gly845Ser mutation, located in exon 22 which is part of CBS2 domain belonging to the cytoplasmic C-end of CLC-1. This missense substitutes a neutral sidechain with a polar one, nevertheless Gly845Ser mutant channels did not show any significant difference with WT ClC-1. Even so, Bennetts and colleagues [26] as a result of mutagenesis experiments highlighted this region as the putative site for ATP binding and possibly involved in the modulation of the common gating [27]. Though placed just aside of the residues directly binding ATP, Glycine 845 seems not to be implicated. Thus results show p.Phe167Leu to be in a dominant form justifying the relative benign clinical presentation of the woman [13,28]. Overall this study has resulted in the addition of 12 new diseasecausing CLCN1 mutations in the list of pathogenic chloride channel mutations, emphasizing the high genetic heterogeneity of chloride channel myotonia where mutations may occur throughout the coding region of CLCN1 yet clustering in hot-spots for dominant-negative interactions. Future investigations of larger familial trees of affected patients, carrying the mutations presented in this work, would confirm evident and reliable correlations between genotypes and phenotypes, for the purpose of designing proper treatment strategies. Conflict of interest All authors of this manuscript declare that they have no conflict of interest. Acknowledgments Special thanks to the “Associazione Amici del Centro Dino Ferrari” for their support. The Telethon Genetic Biobanks Network GTB07001E and the Eurobiobank project are gratefully acknowledged. Our gratitude has to be expressed to the patients for participating in research and for their kindly collaboration. Thanks also to the “Fondazione Malattie Miotoniche”, I.R.C.C.S. Policlinico San Donato.

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