Abstract
Myotonia, defined as delayed relaxation of muscle after contraction, is seen in a group of genetic disorders that includes autosomal dominant myotonia congenita (Thomsen's disease) and autosomal recessive myotonia congenita (Becker's disease).Both disorders are characterized electrophysiologically by increased excitability of muscle fibers, reflected in clinical myotonia. These diseases are similar except that transient weakness is seen in patients with Becker's, but not Thomsen's disease. Becker's and Thomsen's diseases are caused by mutations in the skeletal muscle voltage-gated chloride channel gene (CLCN1). Genetic screening of a panel of 18 consecutive myotonia congenita (MC) probands for mutation in CLCN1 revealed that a novel Gln-68-Stop nonsense mutation predicts premature truncation of the chloride channel protein. Four previously reported mutations, Arg-894-stop, Arg-338-Gln, Gly-230-Glu, and del 1437-1450, were also noted in our sample set. The Arg-338-Gln and Gly-230-Glu mutations were found in patients with different phenotypes from those of previous reports. Further study of the Arg-338-Gln and Gly-230-Glu alleles may shed light on variable modes of transmission (dominant versus recessive) in different families. Physiologic study of these mutations may lead to better understanding of the pathophysiology of myotonia in these patients and of voltage-gated chloride channel structure/function relationships in skeletal muscles.
NEUROLOGY 1996;47: 993-998
Myotonia is a sign of skeletal muscle disease characterized by delayed relaxation of muscle after voluntary contraction or mechanical stimulation. The nondystrophic myotonias usually include three categories of disorders: myotonia congenita, paramyotonia congenita, and hyperkalemic periodic paralysis. Clinical electrophysiologic studies show repetitive electrical discharges of muscle fibers (myotonic runs) in patients with these diseases.
Myotonia congenita (MC) includes two distinct types. Thomsen's disease is an autosomal dominant disorder with onset in childhood and manifested by prominent myotonia [1]; the myotonia can be reduced by muscle activity, a phenomenon termed "warm up." Becker's disease is a similar myotonic disorder with a warm up phenomenon but is transmitted as an autosomal recessive trait. [2] Furthermore, patients with the Becker form of MC frequently have fluctuations in muscle strength.
In human skeletal muscles, almost 80% of total resting membrane conductance is contributed by chloride ions. [3] Hyperexcitability and myotonia can be caused by low chloride conductance of skeletal muscle membranes. [4] Both can be induced by using monocarboxylic aromatic acid compounds (like 9-anthracene-carboxylic acids) and other chemical agents to block chloride ion conductance. [5-9] The skeletal muscle chloride channel (CIC-1) encoded by the CLCN1 gene is the major skeletal muscle voltage-gated chloride channel [10-12] and is genetically linked to myotonia congenita. [13-15] CLCN1, located on chromosome 7q35, [14] is composed of 23 exons. The full-length CLCN1 cDNA is about 3 kb in length and encodes twelve putative transmembrane domains. [16] Mutations in CLCN1 have been shown to cause MC. [10,16-21] In this study, we searched for novel CLCN1 mutations in 18 consecutive probands with the diagnosis of autosomal dominant or recessive MC.
Materials and methods.
Identification of patients.
A total of 18 probands was included in this study. All patients underwent full neurologic examination with particular attention to the neuromuscular system. All probands underwent a standard EMG examination. The diagnosis of Thomsen's MC was made in patients with the following features: prominent musculature, myotonia that warmed-up with use, and the absence of fixed weakness or episodes of transient weakness. Patients were diagnosed as having Becker's MC based on the following criteria: myotonia that warmed-up with use and weakness that improved with muscle use. Occasionally, fixed weakness was present in these patients with Becker's MC. The mode of transmission in families of the probands was established whenever possible by examination of family members.
PCR amplification of patient and control DNA.
Intronic sequence was used to design primers that allowed amplification of CLCN1 exons from DNAs in the sample set of myotonia patients. [21] The polymerase chain reaction (PCR) mixture of total 10 micro liter volume contained: 50 ng genomic DNA; 6.7 micro liter dH2 O; 0.6 micro liter dNTP (125 micro Meter of each deoxynucleoside triphosphate); 0.3 micro liter (25 pmol/micro liter) forward and reverse primer; 1 micro liter reaction buffer (500 mM KCl, 100 mM Tris-HCl, 1.5 mM MgCl2, 0.01% gelatin); 0.25 unit Taq DNA polymerase (5 units/micro liter, Perkin Elmer Corp, Norwalk, CT); 0.1 micro liter [alpha-sup 32 P]-dCTP (Amersham Life Science). The mixtures were initially denatured at 94 degrees C for 3 minutes followed by 30 cycles each of 30 seconds at 94 degrees C, 30 seconds at annealing temperature, and 30 seconds for extension at 72 degrees C.
Single-strand conformation polymorphism (SSCP) analysis.
SSCP was carried out using methods as previously described. [22] In short, the PCR products were diluted and denatured in 50 micro liter of 0.1% SDS/10 mM EDTA. The mixtures were heated at 94 degrees C for 3 minutes after adding loading dye. A total 6 micro liter of mixture was loaded and electrophoresis was performed through 5% non-denaturing polyacrylamide gels at 40 W for 3 to 7 hours. The gels were run under two conditions: room temperature with glycerol and 4 degrees C without glycerol. Gels were transferred to filter paper, dried on a vacuum slab dryer for 1 hour at 85 degrees C, and exposed to x-ray film at -20 degrees C for 12 to 24 hours.
Denaturing sequencing gels.
For detection of small deletions or insertions, denaturing sequencing gel analysis was conducted. The same samples (see SSCP analysis) were loaded on 7% sequencing gels and electrophoresis was performed at room temperature at 80 W for 3 hours. The gels were transferred to filter paper and exposed to x-ray film as above.
Sequencing of the abnormal DNA products.
Ten micro liter of eluted DNAs from the aberrant bands cut from SSCP or sequencing gels were reamplified using the pairs of primers composed of both the original PCR primers and the additional M13 sequencing primer tails (details have been previously described [23]). Samples were purified by a centrifugation wash with a centricon-100 column (Amicon) and sequenced on an Applied BioSystems model 373A DNA sequencer using the dideoxy termination method.
Results.
Screening DNA samples from 18 myotonia congenita patients, we found one novel nonsense mutation, one known nonsense mutation, two known missense mutations, a previously reported common 14 base-pair deletion, and two novel polymorphisms Figure 1 and Table 1.
Figure 1. Positions of voltage-gated chloride channel (ClC-1) myotonia congenita mutations and polymorphisms found in this sample set. Amino acids are denoted by their one-letter code: Q = glutamine, T = threonine, W = tryptophan, G = glycine, R = arginine; two nonsense mutations are shown (stop); Delta 1437-1450 is a 14 base-pair deletion.
Table 1. Chloride channel mutations in myotonia congenita patients
Clinical findings.
All of the patients diagnosed as having Thomsen's MC have prominent musculature, myotonia that warms-up with use, and no weakness. In addition, each of these probands has a family history of MC segregating as an autosomal dominant trait with high penetrance. Five such probands were identified. Eight probands met criteria for diagnosis as having Becker's MC. All patients diagnosed as having Becker's MC have the warm-up sign and all but one have transient weakness. One patient (K2169, #15493) denied transient weakness and had no evidence of weakness on exam. However, this patient was not examined after a period of rest, the condition in which transient weakness is most commonly seen in Becker MC patients. In kindred 1959, re-examination of the family failed to find clinical or EMG evidence of myotonia in the asymptomatic parents or siblings. The remaining five patients have prominent musculature and myotonia with warm-up but insufficient clinical detail was available to classify them further.
Nonsense mutations.
A novel C-202-T mutation in exon two of CLCN1 results in a stop codon in the region close to the amino terminus. This mutation was found in a patient with Becker's MC Figure 2. The result of this nucleotide change will produce a short peptide of 67 amino acid residues without any putative transmembrane domains. Clearly, it will not form a functional chloride channel. Interestingly, the C-2680-T mutation discussed below (causing a stop codon near the carboxyl terminus) was also detected in this patient (see below), suggesting two different mutant alleles causing this patient's autosomal recessive phenotype. Twenty-one DNA samples from normal controls have been screened for this C-202-T mutation by SSCP and no similar aberrant pattern has been observed.
Figure 2. A nonsense mutation was found in a patient with recessive myotonia (#17906, K2288). (A) SSCP gel analysis shows the abnormal band (arrow). Twenty-one normal DNA samples have been investigated and none of them show the similar aberrant band (data not shown). (B) Sequencing data demonstrate the nucleotide change at position 202 is a C-to-T, which results in a stop codon (UAA). Filled arrows point to the position of nucleotide substitution.
Abnormal bands from five samples were noted when SSCP was performed on samples amplified with primer set D23A (which amplified a part exon 23). After aberrant bands were cut from the gel, DNAs were eluted and sequenced. Sequencing data showed a C-to-T transition at position 2680 of partial cDNA sequence of the human CLCN1 Figure 3. When read in frame, this results in an Arg-to-Stop change in the carboxyl terminal (see Figure 1). The predicted protein would be 893 amino acid residues in length. Of these five patients, one is homozygous for this mutation and no other affected individual was noted in this patient's family. This patient and three others were diagnosed as having Becker's disease, while the fifth patient carried the clinical diagnosis of sporadic hyperkalemic periodic paralysis. Eighty-six unrelated normal control samples were investigated and none showed a similar aberrant pattern using SSCP gel analysis. This mutation is of particular interest since it was previously noted in two families with Thomsen's disease, [17] suggesting that the same mutation may present with autosomal dominant or recessive disease inheritance.
Figure 3. SSCP gel analysis of exon 23 of the CLCN1 from myotonia patients. (A) SSCP gels run at room temperature with glycerol. The abnormal SSCP conformers are marked by the arrow. These aberrant bands were noted in samples #13496 (K1959), #17906(K2288), #13826(K2013), #18886(K2475), and #15493 (K2169). Eighty-six unrelated normal control samples have been investigated and none of them show the similar abnormal pattern (data not shown). (B) Sequencing from the abnormal and normal bands on room temperature gel with glycerol show the nucleotide change is C-2680-T. Filled arrows mark the position of nucleotide substitution.
The Arg-338-Gln and Gly-230-Glu mutations can be manifested as either dominant or recessive traits.
We detected an abnormal band in the SSCP pattern in exon nine of the CLCN1 from patients with autosomal dominant MC. One branch of this family is shown in Figure 4. The disease phenotype is highly penetrant in this family. Sequencing of DNA from the abnormal bands showed a missense mutation (G-1013-A) that results in a change of Arg-to-Gln at amino acid position 338. This amino acid change occurs in the intracellular loop between putative transmembrane segments six and seven and has been reported to be responsible for recessive MC. [17]
Figure 4. SSCP analysis of exon nine in kindred 1750. The pedigree shows one branch of this family and the affected individuals were labeled as filled squares (male) or circles (female). All members in this figure were examined by EMG. Primer pair D7 amplifies exon nine, which encodes amino acid residues between the 6th and 7th putative transmembrane dominant. The aberrant bands (arrow) from SSCP gel were sequenced and showed a nucleotide change at position 1013 (G-to-A) of the CLCN1 cDNA. The predicted Arg-338-Gln change was inherited from father by two sons and one daughter. None of the unaffected individuals had this aberrant band. Both clinical and genetic data support that the inheritance in this family is dominant.
A glycine to glutamic acid mutation at amino acid position 230 was previously described in three families with highly penetrant autosomal dominant MC. [18] Using SSCP, we have identified this mutation in one patient with recessive MC. In Figure 5 (D3 primer set) the aberrant SSCP band is shown in the patient's DNA (#13496, kindred 1959) and was also detected in his asymptomatic mother and sister. When sequenced, DNA from the band showed a G to A transition at position 689 of cDNA sequence, which predicts the same amino acid change as reported by George et al. [18] in autosomal dominant MC patients. This same family is segregating the exon 23 nonsense mutation described above. It is seen in the proband and his clinically and electrophysiologically normal father and sister (see Figure 5, D23A). The patient (#13496) is the only member of this family with two mutant alleles. Unlike the pedigrees previously reported, [18] the Gly-230-Glu mutation in this family appears to require a mutation on the second allele to manifest MC. The unaffected parents of the proband were studied both clinically and electromyographically and had no evidence of muscle disease.
Figure 5. SSCP analysis of exon 5 and exon 23 in kindred 1959. The pedigree shows the affected son as a filled square. All other family members were examined by one of the authors (R.C.G.) and underwent EMG. Their examination and EMGs were all normal. Primer pair D3 amplifies exon 5 and primer pair D23A amplifies the 5 prime end of exon 23. The aberrant bands in D3 and D23A primer pairs represent the mutations of G-689-A and C-2680-T, respectively. The G-689-A mutation (empty arrow) was inherited from the mother and the C-2680-T (filled arrow) from the father. Among three children only the boy had both mutations and was diagnosed as having recessive myotonia congenita. The data suggest that both mutations in CLCN1 are responsible for recessive myotonia in this family.
Deletion.
A common deletion of 14 bases in the position of 1437-1450 has been reported. [19] Our investigation of the same region of exon 13 between putative transmembrane domains nine and ten revealed this deletion in a DNA sample from one patient with recessive myotonia congenita (data not shown).
Polymorphisms.
A Trp-to-Gly change predicted by the point mutation of T-352-G in exon 3 of CLCN1 has been found in one patient. The results were compared with 40 normal DNA samples. The same aberrant conformer was noted in five control samples. This polymorphism is in the region of the first putative transmembrane helix.
A C to T nucleotide change at position 261 in exon two was also noted in one patient (K1959, #13496) diagnosed as having recessive MC. This change occurs at the third position of a codon for a threonine residue. The nucleotide change does not alter this threonine. The same polymorphism was noted in 13 of 40 (32%) normal controls.
Discussion.
Molecular analysis of 18 MC probands is reported. Two novel polymorphisms were identified and mutations were found in seven (one dominant, six recessive) of the 18 probands. One novel mutation (Gln-68stop) was identified in a patient with Becker's MC. The previously reported nonsense mutation Arg-894-stop, is a common mutation in our sample set from patients with recessive MC. This mutation predicts a truncated protein lacking the last 95 amino acids of the carboxyl terminus. Putative recognition sites for protein kinase C (PKC) and cGMP-dependent protein kinases are eliminated by this nonsense mutation. A previously recognized deletion (del 1437-1450) was found in one patient. [16] Mutations could not be identified in some of the samples. This may result from insufficient sensitivity of SSCP. Alternate mutation analysis strategies will be pursued with the goal of finding other CLCN1 mutations. Alternatively, the MC phenotype may be caused by another gene in some patients. Unfortunately, insufficient family material is available to test these families for linkage to CLCN1.
Two previously reported mutations are noted in our sample set in patients with different phenotypes from those in the original reports. One of these (Gly-230-Glu), previously seen in three families segregating the Thomsen's phenotype as an autosomal dominant trait, [18] was found in kindred 1959 and segregates as a recessive trait in this patient with Becker's MC. The other (Arg-338-Gln), previously seen in a patient with the Becker's phenotype and apparently recessive, [17] is seen in kindred 1750, a family segregating an allele for the Thomsen's phenotype as an autosomal dominant trait with high penetrance. One possible explanation of this phenomenon is that both Gly-230-Glu and Arg-338-Gln are dominant mutations with incomplete penetrance. However, the identification of both mutant alleles in the patients with recessive disease correlates with the Becker's phenotype seen in those patients. Four unaffected individuals from one of these families (kindred 1959) carry a single mutant allele Figure 5 but were completely normal when studied by careful neurologic and EMG examinations. Furthermore, when these mutations were recognized in patients with the Becker's phenotype, another mutation was found on the second CLCN1 allele.
The reason for variable modes of transmission for a single mutation (Gly-230-Glu or Arg-338-Gln) is not known but comparison of DNA from patients with a mutation that causes different phenotype and transmission in different families may lead to better understanding of this phenomenon. If such a difference can be identified (a second mutation in a single allele, for example), then physiologic study of these mutations in a heterologous expression system may lead to better understanding of the molecular basis of the variable modes of transmission.
Additionally, the same mutations identified in the families with different transmission modes raises an important clinical distinction between Thompsen's disease and Becker's disease. Correlation of the Becker's MC phenotype (i.e., presence of transient weakness) and autosomal recessive transmission may be used as a diagnostic clue in the clinical setting.
Identification of these mutations now makes molecular testing possible in patients. In addition, this knowledge may help to unravel the molecular basis of MC and open new avenues of experimentation for studying this channel and these diseases. Comparative studies of mutations Arg-338-Gln and Gly-230-Glu in the families with different transmission modes may be particularly interesting and lead to better understanding of this phenomenon and of voltage-gated chloride channels in general. Eventually advances in the field may lead to improved clinical treatments.
Acknowledgments
The authors appreciate referral of one patient sample by Dr. Alan Pestronk and helpful discussions with Dr. Kevin Flanigan and with Launce G. Gouw. The authors also thank the DNA core facility at the University of Utah and Mr. Kevin Hurst and Mrs. Wendy Bahr for technical assistance on this project.
- Copyright 1996 by Advanstar Communications Inc.
REFERENCES
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