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Heterozygous CLCN1 mutations can modulate phenotype in sodium channel myotonia A. Furby a,b,⇑, S. Vicart c, J.P. Camdessanche´ a,b, E. Fournier c,d,e, S. Chabrier b,f, E. Lagrue g, C. Paricio b,h, P. Blondy c,e,i, R. Touraine b,j, D. Sternberg c,e,i, B. Fontaine c,e a CHU Saint-Etienne, Hoˆpital Nord, Department of Neurology, Saint-Etienne F-42055, France Rhoˆne-Alpes Reference Center for Rare Neuromuscular Diseases, Saint-Etienne F-42055, France c Assistance Publique-Hoˆpitaux de Paris, National Reference Center for Neuromuscular Channelopathies, Hoˆpital Pitie´-Salpeˆtrie`re, Paris 75013, France d Universite´ Pierre et Marie Curie-Paris VI, Department of Physiology, 75005 Paris, France e CNRS-INSERM-UPMC UMR 1127-7225, Institut Cerveau Moelle, Hoˆpital Pitie´-Salpeˆtrie`re, Paris 75013, France f CHU Saint-Etienne, Hoˆpital Bellevue, Department of Paediatric Physical Medicine and Rehabilitation, Saint-Etienne F-42055, France g UMR Inserm U930, Universite´ Francßois Rabelais de Tours. CHRU de Tours, Service “Neurope´diatrie et Handicaps”, Tours 37044, France h CHU Saint-Etienne, Hoˆpital Nord, Paediatric Intensive Care Unit, Saint-Etienne F-42055, France i Assistance Publique-Hoˆpitaux de Paris, Service de Biochimie Me´tabolique, Centre de Ge´ne´tique, Groupe Hospitalier Pitie´-Salpeˆtrie`re Charles Foix, Paris 75013, France j CHU Saint-Etienne, Hoˆpital Nord, Department of Genetics, Saint-Etienne F-42055, France b

Received 1 May 2014; received in revised form 17 June 2014; accepted 23 June 2014

Abstract Nondystrophic myotonias are characterized by muscle stiffness triggered by voluntary movement. They are caused by mutations in either the CLCN1 gene in myotonia congenita or in the SCN4A gene in paramyotonia congenita and sodium channel myotonias. Clinical and electrophysiological phenotypes of these disorders have been well described. No concomitant mutations in both genes have been reported yet. We report five patients from three families showing myotonia with both chloride and sodium channel mutations. Their clinical and electrophysiological phenotypes did not fit with the phenotype known to be associated with the mutation initially found in SCN4A gene, which led us to screen and find an additional mutation in CLCN1 gene. Our electrophysiological and clinical observations suggest that heterozygous CLCN1 mutations can modify the clinical and electrophysiological expression of SCN4A mutation. Ó 2014 Elsevier B.V. All rights reserved.

Keywords: SCN4A; CLCN1; Myotonia; Nondystrophic myotonia; Myotonia congenita; Paramyotonia congenita; Sodium channel myotonia; Electromyography

1. Introduction Nondystrophic myotonias (NDM) are usually divided in two groups: myotonia congenita (MC) (MIM 160800; ⇑ Corresponding author at: CHU Saint-Etienne, Hoˆpital Nord, Department of Neurology, Saint-Etienne F-42055, France. Tel.: +33 477127622. E-mail address: [email protected] (A. Furby).

http://dx.doi.org/10.1016/j.nmd.2014.06.439 0960-8966/Ó 2014 Elsevier B.V. All rights reserved.

255700) caused by mutations of the CLCN1 gene and paramyotonia congenita (PMC) (MIM168300) or sodium channel myotonia (SCM) (MIM 608390) caused by mutations of the SCN4A [1–5]. MC is transmitted with either a dominant or a recessive inheritance, while PMC and SCM are characterized by allelic autosomal dominant inheritance [3]. Clinical and electrophysiological phenotypes of these disorders have been well described

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and data strongly suggest a correlation between phenotype and genetic defect even if some variability has been observed [1,3,5–7]. No patient with concomitant mutations in both genes has been reported yet. We report herein five patients showing NDM with both chloride and sodium channel mutations and describe for the first time the electrophysiological and clinical phenotypes associated with this genetic setting.

2.1. Case reports

Ophthalmic examination revealed strabismus and eyelid myotonia. Serum creatine kinase (CK) was 1447 U/L (normal < 190). A second needle EMG detected spontaneously abundant myotonic discharges but neither potentiation nor decrement. SET showed a significant decrease of compound muscle action potentials (CMAP) in amplitude (42.3%) and in area (27.3%) immediately after exercise while long exercise tests did not (type II pattern). Sequencing of nine exons of SCN4A revealed a heterozygous mutation in SCN4A: c.3917 G > A p.Gly1306Glu (G1306E, exon 22). This mutation was causative, as it had been described in myotonia permanens phenotype [10]. However, genetic findings were not consistent with the type II ENMG pattern. Therefore we performed sequencing of all exons and flanking intronic regions in CLCN1 and found a heterozygous c.1453 A > G p.Met485 Val (M485V, exon 13) recessive mutation (Fig. 1). Six-hundred milligrams mexiletine treatment dramatically improved myotonia and SET decrement (type III pattern). The patient was the father of two monozygotic male twins. They were born at term after uneventful pregnancy and delivery. Neurologic and general examinations at birth were normal. Intermittent stridor appeared after a few days of life. The attacks became progressively more frequent (about 10 times a day) and more intense. Initial stridor was followed by laryngospasm, general stiffness, and cyanosis but no loss of consciousness. These episodes lasted a few seconds and were electively triggered by cold, bottle-feeding, or esophageal reflux, but could also occur spontaneously. The parents described also rarer transient episodes of tongue or palpebral myotonia. Examinations of the children were always normal between attacks, except for a general muscular hypertrophy that appeared in their first year of life. At 6 months of age, needle examination of twin tibialis anterior muscles detected insertional and spontaneous abundant myotonic discharges. Genetic analysis displayed the same SCN4A and CLCN1 mutations as their father. As there were no lifethreatening or prolonged events, no treatment was started.

2.1.1. Patient 1 In the first days of life, this Caucasian patient showed attacks of laryngospasm and episodic stridor when he was bottle fed. The parents had observed hand contractures and difficulties to open his eyes when he cried. At age 2, clinical examination showed generalized muscle hypertrophy and myotonia in face and limbs. Initial ENMG displayed prolonged and abundant myotonic discharges in vastus and deltoidus muscles. His brother and parents were clinically unaffected and the parents were not related. He had no other major medical problems. At age 26, he regularly practiced sport but myotonia persisted, aggravated by cold temperature. He noticed a warm-up phenomenon. After prolonged exercise, he also experienced stiffness and swallowing problems. He had no weakness and no more hypertrophy.

2.1.2. Patient 2 This 13-year-old child born in Chile lived in France since age 6. We had no medical information about his family. His adoptive parents observed faintness episodes with tachycardia, sweating, and nausea after intakes of sugary foods. He had swallowing difficulties. He experienced stiffness when starting to walk and observed a warm-up phenomenon. Stiffness could also appear after long exercise, with myalgia, especially after cycling or writing. Symptoms were not exacerbated by cold exposure. He never experienced episodes of muscle weakness. Examination showed spontaneous myotonia of hands and periorbital muscles, which did not worsen with repetitive exercise, as well as a slight weakness of interossei muscles. Needle EMG detected myotonic discharges. SET, in warm temperature or after cooling,

2. Methods Electroneuromyograms (ENMG) with exercise tests at room temperature and cold were carried out according to the standardized ENMG protocol previously described [6,7]. It comprised short and long exercise tests and needle examination. Short exercise test (SET) has been shown to be a reliable biomarker to distinguish between SCN4A and CLCN1 mutations [6–9]. Molecular testing was performed after informed consent of the patients or their parents, accordingly to bioethics laws. We followed the algorithm used in our laboratory for sample analysis from patients showing symptoms compatible with SCN4A mutations. The first line investigation consists in sequencing 9 exons of SCN4A (exons 9, 12, 13, 18, 19, 21, 22, 23 and 24), those exons are hotspots for mutations of PMC, SCM and periodic paralyses. The second line investigation depends on clinical and ENMG data: a phenotype compatible with MC, a type II pattern on SET, and a probable recessive mode of inheritance lead to CLCN1 sequencing (all exons and flanking intronic regions); a typical clinical PMC or SCM phenotype, a type I or III pattern on SET, and a probable dominant mode of inheritance lead to sequencing of the 15 remaining exons of SCN4A. We use a step by step approach: when the first line investigation is negative or gives a result that does not fully explain the clinical or electromyographic phenotype, the next line of investigation is then performed.

A. Furby et al. / Neuromuscular Disorders 24 (2014) 953–959 SCN4A

CLCN1

c.3917G>A p.Gly1306Glu

c.1453A>G p.Met485Val

c.4010G>C p.Arg1337Pro

c.803C>T p.Thr268Met

955

Patient 1

T-, F T-, R

P1, F

P1, R

Patient 2

T-, F

T-, R

P2, F

P2, R c.2079T>G p.Ile693Met

c.2926C>T p.Arg976*

Patient 3

T-, F

T-, R P3, F

P3, R

Fig. 1. Electrophoregrams showing the SCN4A and CLCN1 mutations: T-: negative control; P1: patient 1; P2: patient 2; P3: patient 3; F: forward sequencing; R: reverse sequencing. The visualization of electrophoregrams is made in Seqscape software (Life technologies). The same color codes are used for forward and reverse sequences.

and long effort tests pointed to a type II pattern (decrement in amplitude: 69.8%; decrement in area: 57.9%) (Fig. 2). The first line of molecular genetic investigation revealed a new heterozygous missense mutation in SCN4A gene: c.4010 C > G p.Arg1337Pro (R1337P, exon 22). Since SCN4A mutations are not likely associated with type II pattern, CLCN1 gene was sequenced and revealed a heterozygous c.803 C > T p.Thr268Met mutation (T268M, exon 9) (Fig. 1). This mutation was reported several times in literature with a recessive or semi-dominant trait [8,11].

2.1.3. Patient 3 Since early childhood this 25-year-old man showed difficulties opening his eyes when exposed to cold temperature. When he began to walk as a toddler, his parents noticed blockages of the legs that could lead to falls. He also had blockages in hands, with difficulties buttoning up his clothes or using a fork and knife. Abdominal muscles were as well impaired. He experienced chewing difficulties and a slight intermittent strabismus. Early on, he had observed a warm-up phenomenon and these symptoms were exacerbated by

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Fig. 2. Repeated short exercise test performed at cold in patient II with the SCN4A R1337P channel mutation and the CLCN1 T268M channel mutation. Recordings of the abductor digiti minimi CMAP before and after each of the three exercises (noted as 1, 2, 3). Top trace: pre-exercise recording. Below: post-exercise recordings at different times after exercise completion, as indicated left to the tracings. Note the transient decreased CMAP amplitude after exercise that reduced when repeating exercise, 59%, 50%, 25% respectively (pattern II). Scale between two dots: 5 mV, 5 ms.

cold exposure. However he never experienced paralysis. The symptoms worsened from age 12. His parents were not related but came from the same region of Algeria and his father had similar muscular symptoms. Upon examination he showed generalized muscular hypertrophy. He had handgrip, jaw, and eyelid myotonias which improved with warming up. There was a lid-lag sign. There were neither retractions nor weakness. The needle EMG detected abundant myotonic discharges, and short as well as long effort tests were normal, in favor of a type III pattern (decrement in amplitude: 13.8%; decrement in area: 16.4%). The first line study revealed a heterozygous c.2079 T > G p.Ile693Met (I693M, exon 13) new mutation in SCN4A. A mutation (I693T) at the same codon had already been reported in a patient with a peculiar PMC-like phenotype [12,13]. We believed that this I693M mutation was probably causative for the type III electrophysiological profile and the SCM phenotype in our patient. However, even if a warm-up phenomenon can be seen in SCM, the great improvement of stiffness after warm-up and legs involvement in this patient prompted us to continue the genetic investigations in CLCN1 gene which revealed a heterozygous c.2926 C > T p.Arg976* (R976X, exon 23) mutation (Fig. 1). Mexiletine treatment significantly improved myotonia.

3. Discussion All adult patients had clinical traits pointing to possible sodium channel myotonia. Facial myotonia (patients 1, 2 and 3), as well as sensibility to cold (patients 1 and 3) or worsening after prolonged exercise (patients 1 and 2)

were consistent with PMC or SCM. However, myotonia in both legs and a marked warm-up phenomenon were more evocative of MC [1,3,5]. Warm-up phenomenon also occurs in SCM but seems to be less frequent than in MC and depends on underlying mutations. It has not been documented with the specific SCN4A mutations we found [1,3,5,14,15]. In our experience a great improvement of stiffness after warm-up is suggestive of MC. Other clinical symptoms were common to sodium and chloride channel mutations [1]. Strabismus was noticed in patients 1 and 3. Only rare similar cases have been reported in patients diagnosed with MC [16]. In all patients needle EMG showed abundant myotonic discharges, as commonly observed in chloride or sodium channelopathies [6] (see Table 1). However, provocative effort tests in patients 1 and 2 showed a type II pattern which strengthened the hypothesis of a chloride channel involvement (Fig. 2). Type II pattern is highly, although not exclusively, specific for chloride channel myotonias. Hundred percent of the recessive and 71% of the dominant MC patients displayed pattern II after combination of tests performed at room temperature and cold [7]. Moreover, the area decrement was 27% in the first case and 57.9% in the second case which, according to Tan et al. [9], is specific to the type II pattern. In patient 3, the provocative tests showed a type III pattern, in this case the mixed clinical presentation led us to perform CLCN1 sequencing. Our fist line investigation (sequencing of 9 hotspot exons of SCN4A) revealed a mutation in all three patients. There are convincing arguments for the pathogenicity of SCN4A mutations (G1306E, R1337P and I693M) in our patients. Indeed, these mutations belong to highly conserved amino acid sequences. They are located in critical functional regions of the sodium channel. G1306E and R1337P are respectively located in the N-terminal and Cterminal part of the intracellular III-IV hinged-lid fast inactivation (see Fig. 3). Other mutations at codons 1306, 1310 and 1313 in the same III-IV linker are associated with PMC or SCM [7,10,17–20]. The G1306E mutation has been previously associated with clinically severe nondystrophic myotonia, as myotonia permanens [10] or severe neonatal episodic laryngospasm in neonates [19] with recurrent de novo cases. Our patient 1 had a similar but less severe phenotype than patients reported by Lerche et al. [21] and Colding-Jørgensen et al. [10]. The lack of symptoms in the parents suggests a de novo mutation, although they could not be tested. I693M is located in the DII/S4-S5 intracellular linker (Fig. 3). Mutations in this linker have been shown to enhance activation, causing hyperexcitability, or impair slow inactivation process, causing paralysis episodes, [12,13,22,23]. In patient 3, it may be hypothesized that enhanced activation is predominant, as no paralytic episode was noticed. The associated CLCN1 R976X mutation may have also favored the expression of a myotonic rather than paralytic phenotype. This I693M

A. Furby et al. / Neuromuscular Disorders 24 (2014) 953–959

957

Table 1 Main clinical, electrophysiological and genetic characteristics of the patients. Clinical symptoms

Genetic

Myotonia

Warm-up phenomenon

Myotonia worsened by cold

Myotonic discharges

Pattern

SCN4A

CLCN1

+ + + + + +

+++

II

G1306

M485V

++

II

R1337P

T268M

+++

III

I693M

R976X

Patient I

Face and eyelid Larynx Trunk Pharynx Arms Legs

+++ ++ + + ++ +

+ + + + + + then stiffness after exercise

Patient II

Face and eyelid Larynx Pharynx Trunk Arms Legs

+

+ + + + + + then stiffness after exercise

Face and eyelid Larynx Pharynx Trunk Arms Legs

++

Patient III

Electrophysiology

+ + + +++

+ + + + + +

+ + ++ ++

+ + + 1234 5 + +

++ + + ++ ++

+ + 1 2 3 4+ 5 + +

6

+ + + 12 34 5 + + +

6

+ + + + 12 34 5 + + + +

6

6 COOH

Inter III-IV loop = inactivation lid

NH2

I693M (Nav 1.4)

R1337P (Nav 1.4)

G1306E (Nav 1.4)

R976X (CLC-1)

T268M (CLC-1)

M485V (CLC-1)

B C

E

F G H I J

K L M N O P Q

D

R

NH2

COOH

*

Fig. 3. Location of SCN4A and CLCN1 mutations on Nav1.4 and CLC-1 proteins: patient 1 (red); patient 2 (blue); patient 3 (orange).

mutation had not been reported in literature, but two other missense mutations at the same codon had been published: the I693T mutation causes a peculiar PMC with coldinduced weakness but no stiffness [12], as well as neonatal hypotonia in infancy [24]; the I693L mutation has been

reported as the cause of severe myotonia and school-ageonset paralytic episodes [23]. Since clinical and electrophysiological characteristics did not fit with the phenotype associated with the SCN4A mutations initially identified, we sequenced CLCN1 gene

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and found in all our patients, a second heterozygous mutation. The CLCN1 mutations -M485V and T268Mare recessive pathogenic mutations [8,25], with a possible mild dominant effect for T268M [11]. In case 2, T268M mutation is probably recessive, but the hypothesis of a dominant trait cannot be excluded. The R976X CLCN1 mutation has been reported in a cohort of epileptic cases [26]. It has also been identified in two other MC families in our laboratory, in a recessive context with compound heterozygosity. Plassart-Schiess et al. [27] suggest that incomplete dominance with variable penetrance or expressivity better describe the modes of transmission in some families. In a heterozygous state and in the absence of a second CLCN1 mutation, these mutations would usually be clinically silent. In patients 1 and 2, pattern II on SET strongly suggests that heterozygous M485V and T268M mutations influence the electromyographic phenotype. However this electromyographic expression of a heterozygous CLCN1 mutation may be related to mutation type since we found a pattern III in patient 3. Variable decrements during 10 Hz repetitive nerve stimulation had been previously observed by Michel et al. [8] and Colding-Jørgensen et al. [28] in different MC mutations. Clinically, the presence of CLCN1 heterozygous mutations probably enhanced warm-up phenomenon as well as myotonia in legs in our patients. Patient 1, with well-characterized SCN4A G1306E and CLCN1 M485V mutations, is probably the most convincing case for a modifying effect of the CLCN1 heterozygous mutation on the clinical expression and the electrophysiological SET pattern. On the other hand, in patients 2 and 3, SCN4A mutations (R1337P, I693M) are new and have never been studied functionally. In these cases, comparative clinical and electrophysiological observations of relatives with SCN4A but without CLCN1 mutations were not available. Nevertheless, taken altogether, our observations indicate that the clinical and electrophysiological expression of SCN4A mutations is modulated by the concurrence of a heterozygous mutation in a closely related CLCN1 channel. This is in keeping with experimental recordings of myotonic action potentials showing that myotonic behavior depends strongly on synergy between sodium channel gain-of-function and chloride channel loss-offunction defects [29]. Simultaneous co-occurrence of digenic defects involving CLCN1 mutations was found with maltase acid deficiency [30] and dystrophic myotonia type 2 [31–33]. In Germany’s Ulm centre, there was a significantly higher frequency of CLCN1 R894X mutation in DM2 patients (7%) than in the general population (0.3%) [34]. In our laboratory we found only five cases (the three patients presented here, and two other less documented patients) with mutations in both SCN4A and CLCN1 genes, among 546 index cases diagnosed with clinically and genetically-defined NDM. This percentage might be underestimated, since all our NDM patients were not systematically sequenced in

both genes. Other authors have stressed the interest of sequencing both SCN4A and CLCN1 in order to achieve the molecular diagnosis in NDM [35]. In our experience, the first line investigation (SCN4A sequencing) diagnoses 33% of NDM (92% and 96% of PC and SCM respectively) consistent with clinical and electromyographic data. The sequencing of CLCN1 as a second line investigation diagnoses an additional 55% of NDM cases as MC (14% dominant, 84% recessive), with a typical clinical and electromyographic (type II pattern) presentation. In about 10% of NDM, the SCN4A or CLCN1 mutations did not fit with clinical and electromyographic data. In those cases, it may be worth to sequence the totality of coding and flanking intronic regions in both genes, even though we found a second mutation in only 1% of all NDM cases. These cases highlight the solidity of genotype– phenotype correlations in NDM and the importance to continue further genetic analyses when first genetic results do not fit with the clinical or the electromyographic phenotype. In this situation, electrophysiological tests seem to be particularly relevant, redirecting new genetic analysis. Such complex cases will be diagnosed more easily with generalization of the next-generation sequencing technologies. In our patients, the finding of a heterozygous CLCN1 mutation in addition to a dominant SCN4A mutation may help to understand the clinical expression of the disease, and must be taken into account in genetic counseling. Acknowledgments We are grateful to members of Re´socanaux as well as Association Francßaise Contre les Myopathies (AFM) for fruitful discussions and to JF Vernet for editing assistance. The research leading to these results has received funding from the program “Investissements d’avenir”ANR-10-IAIHU-06. References [1] Trip J, Drost G, Ginjaar HB, et al. Redefining the clinical phenotypes of non-dystrophic myotonic syndromes. J Neurol Neurosurg Psychiatry 2009;80:647–52. [2] Meola G, Hanna MG, Fontaine B. Diagnosis and new treatment in muscle channelopathies. J Neurol Neurosurg Psychiatry 2009;80(4):360–5. [3] Matthews E, Fialho D, Tan SV, et al. The non-dystrophic myotonias: molecular pathogenesis, diagnosis and treatment. Brain 2010;133:9–22. [4] Dunø M, Colding-Jørgensen E. Myotonia congenita 2005 [updated 2011 Apr 12]. In: Pagon RA, Bird TD, Dolan CR et al., editors. GeneReviewse [Internet]. Seattle (WA): University of Washington, Seattle; 1993. [5] Raja Rayan DL, Hanna MG. Skeletal muscle channelopathies: nondystrophic myotonias and periodic paralysis. Curr Opin Neurol 2010;23:466–76. [6] Fournier E, Arzel M, Sternberg D, et al. Electromyography guides toward subgroupsof mutations in muscle channelopathies. Ann Neurol 2004;56:650–61.

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