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Year : 2010  |  Volume : 58  |  Issue : 5  |  Page : 743-746

Novel chloride channel gene mutations in two unrelated Chinese families with myotonia congenita

Department of Neurology, The Children's Hospital of Zhejiang University School of Medicine, Hangzhou, China

Date of Acceptance13-Jul-2010
Date of Web Publication28-Oct-2010

Correspondence Address:
Zhe Feng Yuan
57 Zhugan Xiang, Hangzhou 310003, Zhejiang Province
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0028-3886.72163

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 » Abstract 

Myotonia congenita (MC) is a genetic disease characterized by mutations in the muscle chloride channel gene (CLCN1). To date, approximately 130 different mutations on the CLCN1 gene have been identified. However, most of the studies have focused on Caucasians, and reports on CLCN1 mutations in Chinese population are rare. This study investigated the mutation of CLCN1 in two Chinese families with MC. Direct sequencing of the CLCN1 gene revealed a heterozygous mutation (892G>A, resulting in A298T) in one family and a compound heterozygous mutations (782A>G, resulting in Y261C; 1679T>C, resulting in M560T) in the other family, None of the 100 normal controls had these mutations. Our findings add more to the available information on the CLCN1 mutation spectrum, and provide a valuable reference for studying the mutation types and inheritance pattern of CLCN1 in the Chinese population.

Keywords: Chinese, chloride channel gene mutations, myotonia congenita

How to cite this article:
Gao F, Ma FC, Yuan ZF, Yang CW, Li HF, Xia ZZ, Shui QX, Jiang KW. Novel chloride channel gene mutations in two unrelated Chinese families with myotonia congenita. Neurol India 2010;58:743-6

How to cite this URL:
Gao F, Ma FC, Yuan ZF, Yang CW, Li HF, Xia ZZ, Shui QX, Jiang KW. Novel chloride channel gene mutations in two unrelated Chinese families with myotonia congenita. Neurol India [serial online] 2010 [cited 2022 Aug 15];58:743-6. Available from: https://www.neurologyindia.com/text.asp?2010/58/5/743/72163

 » Introduction Top

Myotonia congenita (MC) is a genetic myopathy and includes Thomsen disease (autosomal dominant disorder) and Becker disease (autosomal recessive disorder). [1] Intial studies on the pathogenesis of MC suggested that the skeletal muscle chloride channel gene (CLCN1) as a strong candidate gene. Subsequent studies proved that mutations in the CLCN1 gene responsible for the two types of human MC. [2],[3] In humans, the CLCN1 gene has been localized to chromosome 7q35, and it contains 23 exons that encode 988 amino acids. [1] Human skeletal muscle chloride channel (CLC-1) is a homodimer with a unique double-barreled molecular structure, it is composed of 18 transmembrane helices. To date, nearly 130 different CLCN1 mutations have been reported, [1] and the mutation spectrum is further expanding. [4] These mutations involved 23 exons and most of them are recessive. Thomsen disease is caused by heterozygous mutations, whereas Becker disease by homozygous or compound heterozygous mutations. There is no clear information regarding the relationship between the genotype and clinical phenotype. [5] Fialho et al.[6] obtained information on more than 300 MC patients and found that exon 8 was a mutational hotspot in Thomsen disease. Most of the MC mutations reported to date have been identified in Caucasian families, and very few mutations have been identified in the Chinese population. [7],[8],[9] In this study, we identified two new CLCN1 mutation sites (A298T and M560T) and one known mutation site (Y261C) in two Chinese families with MC, thus adding further data to the CLCN1 mutation spectrum.

 » Materials and Methods Top

Clinical data

Family 1: Proband 1, a 6.5-year-old girl had been experiencing muscle myotonia and stiffness since five years. These symptoms used to get aggravated under cold conditions and relieved on warming up or after repeated movement. She had mild hypertrophy of the limb muscles, especially in the forearm and gastrocnemius muscle and also percussion myotonia. Serum creatine kinase (CK), electrolytes, and calcium were normal and acetylcholine receptor antibodies were negative. Electromyograpy (EMG) revealed bursts of myotonia. Similar symptoms were observed in the family, grandfather, father, and aunt. They were subsequently proven genetically to have the same type of MC [Figure 1]a.
Figure 1: a: Family 1; the filled symbols indicate affected individuals, and the open symbols indicate unaffected individuals. The arrow points to proband 1.
b: Sequencing results showed that proband 1 had a heterozygous G-to-A transition at nucleotide 892 of exon 8, which led to the conversion of alanine to threonine at amino acid position 298 (A298T). However, no such mutations were observed in the controls.

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Family 2: Proband 2 a 8-year-old girl had been experiencing stiffness since six months. The stiffness was characterized by a slow pace at the commencement of motor activities. The symptoms used to be aggravated under cold conditions or when the patient experienced nervousness, and used to get relieved on warming up or after repeated movement. She had mild limb-muscle hypertrophy, especially in the muscles of the forearm, gastrocnemius, and triangular muscle and also percussion myotonia. Serum CK, electrolytes, and calcium were normal and acetylcholine receptor antibodies were negative. The EMG showed myotonic potentials. The patient's father had mild symptoms of myotonia, which gradually disappeared as he grew older. At present, his neurological examination is normal. Her mother, however, had no similar history, [Figure 2]a.
Figure 2: a: Family 2; the filled symbol indicates affected individuals, and the open symbol with a central black spot indicates the asymptomatic carrier,the arrow points to proband 2.
b: Sequencing results showed a heterozygous A-to-G transition at nucleotide 782 in exon 7 (Y261C) and a heterozygous T-to-C transition at nucleotide 1679 in exon 15 (M560T). The controls, however, showed no similar mutations at either of these sites.

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To assess the possible occurrence of polymorphisms in any detected nucleotide substitutions, we analysed 100 healthy children with the same ethnic background as control, coming from the Children's Hospital of Zhejiang University School of Medicine. The study was approved by the Ethics Committee of the hospital.

Genetic analysis

After obtaining informed consent, a standard DNA extraction method was used to extract genomic DNA from vein blood samples of members of families 1 and 2 and 100 normal controls. Polymerase chain reaction (PCR) and DNA sequencing were used to analyze the mutations in the CLCN1 encoding region. The primers used in the PCR and DNA sequencing of all the 23 exons of the CLCN1 gene were designed by appropriately modifying known primers mentioned in other studies. [10],[11] The PCR reaction mixture (total volume, 50 μl) contained 100 ng genomic DNA, 1.5 mM MgCl2 , 200 μM dNTPs, 20 pmol each of the forward and reverse primers, and 2.5UEx Taq enzyme (TaKaRa) in the reaction buffer. After amplification, the PCR products were separated on 1.5% agarose gel for one hour at 100 V and sequenced by using an ABI3730 system (Bio Basic Inc, Shanghai, China). Exons that showed a mutation in the first round of sequencing were amplified and sequenced again to exclude the mismatching caused by the low fidelity of Taq DNA polymerase. Repeated sequencing was performed to confirm the mutations.

 » Results Top

Direct sequencing of the 23 exons of the CLCN1 gene revealed a heterozygous G-to-A transition at nucleotide 892 of exon 8 in affected members of family 1; this mutation resulted in the conversion of alanine to threonine at amino acid position 298 (A298T). However, no such mutations were observed in the controls [Figure 1]b. DNA sequencing of the members of family 2 revealed compound heterozygous mutations in the genome of the proband 2; these mutations included a heterozygous A-to-G transition at nucleotide 782 in exon 7 that resulted in the conversion of tyrosine to cysteine at amino acid position 261 (Y261C) for one allele, and a heterozygous T-to-C transition at nucleotide 1679 in exon 15 that resulted in the conversion of methionine to threonine at amino acid position 560 (M560T) for another allele. The proband's father and mother had the heterozygous mutations M560T and Y261C. No such mutations were found in the 100 controls. In addition, a known polymorphic mutation D718D was also identified [Figure 2]b.

 » Discussion Top

Incidence studies show a higher incidence of the recessive-type MC disease than the dominant-type. In a study of 142 families of MC in West Germany, dominant-type accounted for 19% and recessive-type for 73%. [6] Similarly, a dominant inheritance pattern was found in only 20% in a study of approximately 130 different mutations of CLCN1. [1] In contrast, in the Chinese literature most of the reported cases (86.1%) had autosomal dominant inheritance pattern. [12] The differences in the incidence rates of the two types of MC in the Chinese and Caucasians may be attributable to the racial differences.

Reports on gene mutations in MC families of Chinese population are rare and even not known in mainland China. We identified two new CLCN1 mutation sites. In the studies on family 1, we found a new heterozygous mutation site A298T in proband 1 and other members of the family. This mutation is a heterozygous G-to-A transition at nucleotide 892 in exon 8, which leads to the conversion of alanine to threonine at amino acid position 298. A298T is located at the junction of H-I helix of the CLC-1, which is the interface of the chloride channel dimer; this junction is recognized as a predilection site of dominant mutations [13] and a hotspot region of dominant MC mutations. [6] A dominant mutation F297S has been found in the region adjacent to A298T. [6] The mutation can drastically shift the channel voltage dependence to more positive voltages through a dominant negative effect (the negative impact of the mutated subunit on the WT subunit), thereby increasing the half-activated voltage (V1/2) in the heteromeric mutant/WT complexes; this phenomenon can significantly reduce CLC-1's open probability and chloride ions conductance at physiological voltages, and thus increase the excitability of the membrane. All three generations of family 1 showed a dominant inheritance pattern of the disease, and genetic studies confirmed that all the patients had an A298T mutation; however, the 100 normal controls did not show similar changes. Thus, we suggest that the A298T mutation is similar to the F297S mutation and can cause myotonia by causing CLC-1 dysfunction through a dominant negative effect. However, the exact impact of the mutation needs to be confirmed by future studies on function.

In family 2, we found a compound heterozygous mutations (Y261C and M560T) in proband 2. The proband's father and mother had the heterozygous mutations M560T and Y261C. The M560T mutation, which has not been reported in China or in any developed countires, is located on the Q helix of the CLC-1. The neighboring mutations include I556N, V563I, and A566T; these mutations have different degrees of impact on channel function. All these mutations exhibit recessive inheritance. The I556N mutation is characterized by incomplete penetrance. [14] The V563I mutation is located in a highly conserved amino acid region, and has an important effect on the pore and the fast gate. [15] Patients with the A566T mutation show mild to moderate myotonia. [6] The Y261C mutation, located on the G helix of CLC-1, was first reported by Mailander et al.[16] The patient had a compound heterozygous mutations (Y150C and Y261C) and typical symptoms of myotonia. The patient's mother (Y261C) had no clinical symptoms, and a muscle EMG did not indicate myotonia. An EMG examination of the patient's father (Y150C) indicated myotonia, although there were no clinical symptoms; these finding suggested potential myotonia. Wollnik et al. [17] used the Xenopus expression system to study these mutations and found that the Y261C mutation had little impact on CLC-1 function, thereby indicating the presence of other mechanisms that are not completely represented by the Xenopus expression system. Proband 2 (Y261C and M560T) showed typical myotonia; the proband's father (M560T) had shown mild symptoms of myotonia in his teens and the mother (Y261C) had no clinical manifestations. This finding suggested that the MC in this family was characterized by a recessive pattern of inheritance, and that the M560T mutation showed incomplete penetrance. Besides the mutation of Y261C, the proband 2 had mutation in M560T. The combined effect of these mutations had a major impact on the functioning of CLC-1, thus resulting in the typical clinical symptoms of myotonia. However, the exact mechanism underlying the occurrence of myotonia needs to be investigated future. In addition, a known polymorphic site D718D was also found in family 2.

 » References Top

1.Lossin C, George AL Jr. Myotonia congenita. Adv Genet 2008;63:25-55.  Back to cited text no. 1
2.Koch MC, Steinmeyer K, Lorenz C, Ricker K, Wolf F, Otto M, et al.The skeletal muscle chloride channel in dominant and recessive human myotonia. Science 1992;257:797-800.  Back to cited text no. 2
3.George AL Jr, Crackower MA, Abdalla JA, Hudson AJ, Ebers GC. Molecular basis of Thomsen's disease (autosomal dominant myotonia congenita). Nat Genet 1993;3:305-10.  Back to cited text no. 3
4.Moon IS, Kim HS, Shin JH, Park YE, Park KH, Shin YB, et al. Novel CLCN1 mutations and clinical features of Korean patients with myotonia congenita. J Korean Med Sci 2009;24:1038-44.  Back to cited text no. 4
5.Colding-Jorgensen E. Phenotypic variability in myotonia congenita. Muscle Nerve 2005;32:19-34.  Back to cited text no. 5
6.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;130:3265-74.  Back to cited text no. 6
7.Jou SB, Chang LI, Pan H, Chen PR, Hsiao KM. Novel CLCN1 mutations in Taiwanese patients with myotonia congenita. J Neurol 2004;251:666-70.  Back to cited text no. 7
8.Kuo HC, Hsiao KM, Chang LI, You TH, Yeh TH, Huang CC. Novel mutations at carboxyl terminus of CIC-1 channel in myotonia congenita. Acta Neurol Scand 2006;113:342-6.  Back to cited text no. 8
9.Burgunder JM, Huifang S, Beguin P, Baur R, Eng CS, Seet RC, et al. Novel chloride channel mutations leading to mild myotonia among Chinese. Neuromuscul Disord 2008;18:633-40.  Back to cited text no. 9
10.Lorenz C, Meyer-Kleine C, Steinmeyer K, Koch MC, Jentsch TJ. Genomic organization of the human muscle chloride channel CIC-1 and analysis of novel mutations leading to Becker-type myotonia. Hum Mol Genet 1994;3:941-6.  Back to cited text no. 10
11.Lehmann-Horn F, Mailander V, Heine R, George, AL. Myotonia levior is a chloride channel disorder. Hum Mol Genet 1995;4:1397-402.  Back to cited text no. 11
12.Zhang YW, Zhang SS, Shang HF. Clinical characteristics of myotonia congenita in China. Neural Regen Res 2008;3:216-20.  Back to cited text no. 12
13.Duffield M, Rychkov G, Bretag A, Roberts M. Involvement of helices at the dimer interface in ClC-1 common gating. J Gen Physiol 2003;121:149-61.  Back to cited text no. 13
14.Plassart-Schiess E, Gervais A, Eymard B, Lagueny A, Pouget J, Warter JM, et al. Novel muscle chloride channel (CLCN1) mutations in myotonia congenita with various modes of inheritance including incomplete dominance and penetrance. Neurology 1998;50:1176-79.  Back to cited text no. 14
15.Sangiuolo F, Botta A, Mesoraca A, Servidei S, Merlini L, Fratta G, et al. Identification of five new mutations and three novel polymorphisms in the muscle chloride channel gene (CLCN1) in 20 Italian patients with dominant and recessive myotonia congenita. Mutations in brief no. 118. Online. Hum Mutat 1998;11:331.  Back to cited text no. 15
16.Mailander V, Heine R, Deymeer F, Lehmann-Horn F. Novel muscle chloride channel mutations and their effects on heterozygous carriers. Am J Hum Genet 1996;58:317-24.  Back to cited text no. 16
17.Wollnik B, Kubisch C, Steinmeyer K, Pusch M. Identification of functionally important regions of the muscular chloride channel CIC-1 by analysis of recessive and dominant myotonic mutations. Hum Mol Genet 1997;6:805-11.  Back to cited text no. 17


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