Note: This article was written in 1997 as a graduate student project. Obviously, the provided information is dated and research has progressed in the meantime. The article is merely posted to provide a brief introduction to the disorder. Language problems were not corrected.

Phenotype of autosomal recessive generalized myotonia:

Fig. 1: Recessive generalized myotonia with muscular hypertrophy, 37 years old (2, p. 70)

The effects of GM are easily demonstrated by applying a short blow to e.g., the rectus femoris muscle. This mechanical stimulus produces a temporary sustained indent at the point of impact - a symptom referred to as percussion myotonia (Fig. 2)(1, 2, 4). Typical for GM is also a transient weakness in newly performed movements, especially after exhaustive exercise (5, 6). Submaximal contractions affected by GM also may involuntarily increase in intensity to close to tetanic levels (7). First signs of GM appear at the age of five and continue to become more distinct until the beginning of puberty (8). Unusual gait, problems with stair climbing, and seemingly awkward movements best describe the onset of the malady. In most cases the symptoms quickly reach a steady state over a few years, albeit with variable degrees of severity. Even though GM doesn't have any direct effects on the patient's life expectancy, complications due to an increased number of accidents caused by motor impediment must be expected (2, p.64).

Fig. 2: Mechanical myotonic reaction (percussion myotonia) in the arm and the thigh (2).

There are several phenotypically similar diseases. The most closely related (disease) is autosomal dominant myotonia (MC), also known as Thomsen's disease (9). Until the early 1970's, GM and MC were combined under the more general term myotonia congenita. Based on the findings of their different mode of inheritance by P.E. Becker (10), GM and MC were recognized as separate entities in 1973. During the course of recent research, GM was found to additionally differ from MC by a later onset and the occurrence of transient weakness after rest (1, 3, 11). It has thus become easier to identify and to distinguish GM from related conditions. In contrast to  paramytonia congenita for example, neither GM nor MC is pronounced under cold conditions. Furthermore, muscular atrophy as it is seen in myotonic dystrophy, has never been observed in any GM or MC patient.
 
 

Physiology of GM:

Contractions in skeletal muscle are elicited by stimuli arriving from the peripheral nervous system. Action potentials travel down the axons of a-motorneurons and reach the neuromuscular junction. Here, the incoming depolarization wave triggers exocytosis of acetylcholine consequently creating an excitatory endplate potential on the postsynaptic side which is translated into an action potential (AP). The AP spreads concentrically across the muscle fiber membrane, i.e., the sarcolemma, and into invaginations thereof. With the help of this so-called t-tubular system, it is ensured that the excitatory message is quickly and evenly distributed along and into the muscle fiber. However, during APs, it has been observed that the t-tubules tend to accumulate positive charge due to the outflux of cellular K⁺. It has been argued that the t-tubular confinement in space leads to actual ion concentration shifts that alter the skeletal muscle membrane potential - an effect easily reproducible by applying the Nernst equation. In normal individual, this effect is attenuated by a high chloride conductance (g_Cl) of the resting membrane. The elevated t-tubular K⁺ concentration is thus electrically neutralized by a simultaneous Cl^- influx. Consequently, any decrement of g_Cl is apt to cause electrical perturbations in the t-tubules which are reported to the cytoplasm as further excitatory stimuli. The following contractions are thereby uncoupled from central excitation control and can be observed as cramps (12).
 
 

Molecular evidence for defects in the human skeletal muscle membrane Cl^- channel in GM:

After GM and MC had been classified as two separate diseases, Iaizzo and colleagues proceeded to elucidate the physiological basis for the symptoms (3). In vitro experiments with simultaneous recording of force development and EMG activity after electrical stimulation demonstrated a high correlation of prolonged electrical after activity and the decay of force in the examined GM muscle (Fig. 3). It was also noted that electrical after activity subsided after repetitive stimulation as contraction times concomitantly leveled out to normal values. Spontaneous electrical activity was observed in some cases, again correlating well with a simultaneous muscle twitch. The same results could be observed in control muscle tissue which had been incubated with 9-anthracene carboxylic acid, a Cl^- channel blocker. Resting membrane potential and intracellular calcium concentration were measured as normal. Therefore, a connection of GM and Cl^- channel dysfunction could be readily assumed.
Fig. 3: Decreased electrical after activity following successive stimulation. Six successive twitches of fiber sements form a patient with GM. The after activity subsequently decreased (3).

In 1991, C. Franke et al. conducted further in vitro experiments using patch clamp techniques on human GM muscle cells (8). Incubation with sodium channel blocking tetrodotoxin (TTX) in a chloride free solution exhibited normal values for potassium conductance. g_Cl however proved to be decreased and was equal to zero in some cases. Since g_Cl contributes 80 % to the normal membrane conductance (13), this observation was interpreted as a strong indicator for involvement of aberrant Cl^- channels in GM. Additionally, Na⁺ channels showed abnormal gating qualities with belated openings and prolonged mean open times, i.e., Na⁺ channel inactivation had to be altered.

In the same year, K. Steinmeyer and his team discovered a genetic defect in the murine ClC-1 gene coding for the skeletal muscle chloride channel in ADR mice (14). Since these animals had previously been suggested to pose an adequate model for GM (15), DNA from homozygous adr lines was subjected to Southern blotting with labeled rat ClC-1 probes. Hybrids with the adr allele were always larger than control hybrids, whereas adr/+ setups demonstrated presence of both hybrid sizes. Theses results pointed toward an insertional mutation. The following Northern analysis with a probe from the 5' end of ClC-1 revealed Cl^- channel mRNAs of variable sizes in adr/adr mice. None of these mRNAs was of the same size as the mRNA detected in the control. In order to determine the type of the adr mutation, a complementary DNA library was screened with ClC-1 cDNAs, yielding clones homologous to the rat cDNA probe at the 5' end but not at the 3' end. Database research strongly suggested an ETn transposon insert into the adr ClC-1 gene which gave rise to various abnormal splice products, i.e., mRNA of the affected ClC-1 gene could not be properly translated. As a result, homozygous ADR mice were deprived of their skeletal muscle chloride channels. These observations were compatible with a mathematical model of GM, which predicted penetrance, if g_Cl was reduced below 20 % of its contribution to total membrane conductance (16, 17). This would explain why mice heterozygous for adr were clinically not affected.

Driven by these results, M.C. Koch and researchers began to hunt for the gene coding for the human skeletal muscle chloride channel (18). Rat ClC-1 was used as a template for homology screening of human DNA which lead to a partial ClC-1 cDNA. The actual gene locus could be determined as 7q32 by utilizing somatic cell hybrid methods (19). Comparison of GM ClC-1 base pair sequences to other chloride channel genes demonstrated the presence of several highly conserved regions. These had previously been identified as putative membrane spanning domains by evaluation of hydrophobicity assays (20). Although in some cases a mutation in one of these regions could be found, patients with no apparent aberration of in the scanned sequences were noted. Hence, further mutations contributing to the effects of GM were postulated. The Koch et al. studies also revealed a linkage of mutated ClC-1 to dominant myotonia, i.e., MC. This was of special interest, since the dominant mode of inheritance gave rise to the conclusion that the structure of the Cl^- channel could be of homomultimeric nature (21).

Fig. 4: Location of mutations in the ClC-1 choride channel protein. A preliminary transmembrane model is shown that is based on results from previous papers (23).

In recent years, it has become clearer and clearer that variable penetrance and alternating mutations of the ClC-1 gene account for the inconsistent phenotype of GM. In 1994, R. Heine et al. conducted PCR amplifications of specific ClC-1 exons from GM patients using intronic primers with subsequent PCR sequencing (22). These examinations uncovered two novel point mutations and one case of a 4 bp deletion. While the point mutations lead to missense products, the latter genetic defect gave rise to a -1 frameshift resulting in a truncated nonsense gene product (Fig. 4). Surprisingly, the patient with the truncated protein showed less severe symptoms of GM than the patients with the missense mutation. This raised the question, whether there could be alternative, so far not detected, muscle chloride conductance pathways in mammalian skeletal muscle. The earlier repor of aberrant Na⁺ gating had already been traced to genetic defects in the gene coding for skeletal muscle Na⁺ channels, located on chromosome 17q23-25 (24) and had so far not been reported in patients diagnosed with GM. Furthermore, in 1991, it had already been established that neither releaser nor reception of acetylcholine were important for the mechanism of myotonia in ADR mice (25). Dysfunctional neuromuscular transmission could therefore be excluded as a possible effector of GM. Contrarily, Birkle et al. described abnormal fatty acid composition in sarcolemma and sarcoplasmic reticulum from the same animal model (26). Such conditions could allow for changes in membrane fluidity thus affecting the function of ion channels. These results were supported by earlier findings of altered membrane characteristics in cells from myotonia patients (27).

Fig. 5: Structure of the human ClC-1 gene. The scheme shows the complete protein-coding region of the ClC-1 gene as open boxes. The first base of the initiator codon (ATG) is given as 1, the last base of the stop codon as 2967. The dashed lines indicated 5' and 3' non-translated regionsof exons. The gray areas represent the regions of the exons that code for the putative transmembrane domains (D1-D12). D13 is a highly conserved cytoplasmic segment near the carboxy-terminal end of the coding region. Where dterminded, the size of the introns is shown above. The sizes of exons are drawn to scale and the first base of the individual exons is denoted by the numbers at the right of the arrowheads.

In 1994, C. Lorenz et al. finally succeeded in supplying complete information on the exon-intron boundaries and flanking intron sequences of the coding region of the human ClC-1 gene (28). Genomic libraries were screened with partial human cDNA which identified three 15 kb clones that together spanned an approximate 40 kb region. The clones were inserted into a Bluescript vector, digested and subsequently hybridized with specific ClC-1 cDNA probes applying Southern techniques. Thereby, the researchers were able to identify 23 exons in the human ClC-1 gene (Fig. 5). In a following experiment, the recombinant plasmids were used to sequence the clones by utilizing the dideoxynucleotide (ddNTP) method. Plasmid specific primers labeled with a ³⁵S-dATP initiated DNA replication in four different setups. Each had a supply of three types of regular deoxynucleotides, while the fourth was replaced by its ddNTP analogue. Upon insertion of the ddNTP, DNA synthesis was terminated, yielding DNA fragments that ended with a specific ddNTP. The four incubations were separated by gel electrophoresis and visualized in an autoradiogram. A line-up of their positions reflected the base pair sequence of the examined clone. It could thus be determined that the human ClC-1 gene consisted of 2964 bp coding for 988 amino acids (Fig. 5) and is most likely subject to a complex transcriptional regulatory mechanism. The latter was suggested after database search conducted on the 5' upstream region indicated the presence of several elements that had previously been proven to play a regulatory role in the transcription of other genes (Fig. 6). Amongst these elements were a TATA box at base position -130 with respect to the start codon, three myogenic MyoD family E-boxes, a CCAC box, and a dodecameric palindromic sequence at -260. Lorenz et al. continued their experiments using intronic primers to amplify specific GM exons of ClC-1, which were examined by single strand conformation polymorphism analysis (SSCA). The amplified GM exons were denatured and run on a gel together with wildtype exon strands. Change of temperature or pH resulted in a different strand conformation if a mutation was present, i.e., a difference in motility could be observed. Abnormal alleles were sequenced and displayed numerous substitutions like F413C, V327I, R496S, and a previously reported 14 bp deletion in exon 13. It is of interest that there was a distinct variability for hosting the different defects, which meant that GM could not be attributed to specific single mutations in ClC-1. Two of the point mutations were further explored by injecting mutant V327I or R496S RNA existing of exons only (cRNA) into Xenopus laevis oocytes and monitoring Cl^- currents via voltage clamping. V327I, a mutation in the vicinity of a splice junction, exhibited typical g_Cl values. Therefore, the genetic defect had be based on abnormal splicing and truncated gene products. R496S on the other hand, was a non-conservative substitution in a highly conserved transmembrane region. No Cl^- currents were measured in this setup. Consequently, oocytes were injected with a 1:1 wildtype/R496S cRNA mixture which still gave a 50 % Cl^- conductance compared to plain wildtype injection. These results were seen as evidence that R496S did not interfere with wildtype channels, but caused a non-functional ClC-1 product in homozygous patients. Hence a homo(tetra)meric Cl^- channel was proposed.

Fig. 6: Sequence of the human ClC-1 5'-untranslated region. (Putative) regulatory elements are typed in bold face. The palindromic sequence encompassing and EcoR1 site is underlined (28).

Two years later, in 1996, Zhang and colleagues conducted a series of similar experiments that added further mutations to the list of GM causing defects (11). One of the more interesting mutations was a G2680T substitution that introduced a premature stop codon after the 893rd amino acid residue. The such truncated protein lacked recognition sites for protein kinase C and GMP dependent protein kinases which disabled its Cl^- gating function. Confusion arose by the discovery of R338Q in a patient suffering from MC. This specific mutation had, until then, been considered to exclusively cause GM. Additionally, an MC specific G230E substitution was found in a GM patient, further complicating the dilemma. Therefore, the generally accepted distinction of GM and MC by mutation had to be reviewed. Zhang et al. made it obvious that a prediction of the phenotype based on the currently known genomic aberrations was not feasible.
 
 

Outlook for future experiments:

Another important issue to be reviewed, is the contribution of other genetic defects like dysfunctional Na⁺ channels or altered membrane conditions to the phenotype of GM. Screening for a additional mutations on 17q23-25 is therefore a need for complete evaluation of GM.

It should also be noted, that the newly evolving techniques of gene therapy and cell therapy could harbor exciting new possibilities for GM research. For example, an interesting approach in not treating a congenital muscular disease but actually curing it, has been undertaken by Peter Law and the Cell Therapy Research Foundation. As described in Lyon's and Gorner's "Altered Fates" (29), the researcher tried to introduce functional dystrophin into the developing muscle of young boys suffering from X-chromosomal muscular dystrophy using the embryonic versions of adult muscle cells, i.e., myoblasts. By injecting large amounts of these myoblasts directly into the muscle tissue, Law hoped to achieve fusion of the such with the muscle fibers. Since a number of muscle genes are turned on in these myoblasts, it is possible that dystrophin is expressed and properly incorporated into the muscle structure. The results of these experiments are still vigorously discussed, but the safety of the procedure has now been established. Hence, it is a feasible option to reconduct these experiments with an animal model, e.g. ADR mice, to determine the value of such treatment for GM. Although, even in the case of marked improvement of the treated muscle, it still remains questionable how to facilitate or even evade individual treatment of each muscle.

Another delivery system has been suggested by geneticist Jon Wolff from the University of Wisconsin at Madison. It has been reported that mouse cells have the ability to merely incorporate injected DNA. However, the DNA does not fuse with the chromosome, but remains an autonomous but concomitantly fully expressed gene in the nucleus. So far, results of marker labeled genes injected into muscle are meager, but promising.

One further possibility of gene therapy uses shell-like carrier cells with receptors specific to skeletal muscle cells. These cells would carry plasmids hosting the ClC-1 gene which could be expressed after entering the target cell. The simpleness of a single infusion into the bloodstream is striking, albeit still a mere vision of research.

Note: The author was diagnosed with GM at the age of 14.
 

 References:

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2. Becker, P.E., Myotonia congenita and syndromes associated with myotonia, 1977, Thieme (Stuttgart)
3. Iaizzo, P.A., Lehmann-Horn, F. (1990). The correlation between electrical after-activity and slowed relaxation. Muscle & Nerve 13: 240-246
4. Dubowitz, V., Muscle disorders in childhood, 1995, 2nd edition, W.B. Saunders (Philadelphia): p. 271
5. personal observation, e.g., after swimming exercise
6. Rüdel, R., Ricker, K. and Lehmann-Horn, F. (1988). Transient weakness and altered membrane characteristic in recessive generalized myotonia (Becker). Muscle & Nerve 11: 202-211
7. personal observation during an leisure time experiment on a force dynamometer with visual display of force development; the author also frequently experienced build-up of severe cramps after exercise upon minor voluntary contraction
8. Franke, C., Iaizzo, P.A., Hatt, H., Spittelmeister, W., Ricker, K. and Lehmann-Horn, F. (1991). Altered Na⁺ channel activity and reduced Cl^- channel conductance cause hyperexcitability in recessive generalized myotonia (Becker). Muscle & Nerve 14:762-770
9. Thomsen, J. (1876). Tonische Krämpfe in willkürlich beweglichen Muskeln in Folge von ererbter psychischer Disposition. Archiv Psychiatrischer Nervenkrankheiten 6: 702-718
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12. George, A.L. (1995). Molecular genetics of ion channel diseases. Kidney International 48: 1180-1190
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14. Steinmeyer, K., Klocke, R., Ortland, C., Gronemeier, M., Jockusch, H., Grüner, S. and Jentsch, T.J. (1991). Inactivation of muscle chloride channel by transposon insertion in myotonic mice. Nature 354: 304-308
15. Watkins, W.J. and Watts, D.C. (1984). Biological features of the new A2G-adr mouse mutant with abnormal muscle function. Laboratory Animals 18: 1-6
16. Adrian, R.H. and Marshall, M.W. (1976). Action potentials reconstructed in normal and myotonic muscle fibres. Journal of Physiology 258: 125-143
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18. Koch, M.C., Steinmeyer, K., Lorenz, C., Ricker, K., Wolf, F., Otto, M., Zoll, B., Lehmann-Horn, F., Grzeschik, K.-H. and Jentsch, T.J. (1992). The skeletal muscle chloride channel in dominant and recessive human myotonia. Science 257: 797-800
19. later determined to be 7q35 in Abdalla, J.A., Casley, W.L., Cousin, H.K., Hudson, A.J., Murphy, E.G., Cornélis, F.C., Hashimoto, L. and Ebers, G.C. (1992). Linkage of Thomsen disease to the T-cell-receptor beta (TCRB) locus on chromosome 7q35. American Journal of Human Genetics 51: 579-584
20. Jentsch, T.J. (1993). Chloride channels. Current Opinion in Neurobiology 3: 316-321
21. Steinmeyer, K., Lorenz, C., Pusch, M., Koch, M.C. and Jentsch, T.J. (1994). Multimeric structure of ClC-1 chloride channel revealed by mutations in dominant myotonia congenita (Thomsen). The EMBO Journal 13(4): 737-743
22. Heine, R., George, A.L., Pika, U., Deymee, F., Rüdel, R. and Lehmann-Horn, F. (1994). Proof a non-functional muscle chloride channel in recessive myotonia congenita (Becker) by detection of a 4 base pair deletion. Human Molecular Genetics 3(7): 1123-1128
23. Meyer-Kleine, C., Steinmeyer, K., Ricker, K. and Jentsch, J.T. (1995). Spectrum of mutations in the major human skeletal muscle chloride channel gene (ClCN1) leading to myotonia. American Journal of Human Genetics 57: 1325-1334
24. Rüdel, R., Ricker, K., and Lehmann-Horn, F. (1993). Genotype-phenotype correlations in human skeletal muscle sodium channel diseases. Archives of Neurology 50: 1241-1248
25. Költgen, D., Brinkmeier, H. and Jockusch, H. (1991). Myotonia and neuromuscular transmission in the mouse. Muscle & Nerve 14: 775-780
26. Birkle, D.L., Shahamat, V., Lucci, J., Johnson, E.A., Riggs, J.E. and Azzaro, A.J. (1993). Abnormal fatty acid composition in sarcolemma and sarcoplasmic reticulum from myotonic ADR mouse muscle. Biochimica et Biophysica Acta 1146: 236-242
27. Rüdel, R. and Lehmann-Horn, F. (1985). Membrane changes in cells from myotonia patients. Physiological Reviews 65(2): 310-356
28. Lorenz, C., Meyer-Kleine, C., Steinmeyer, K., Koch, M.C. and Jentsch T.J. (1994). Genomic organization of the human muscle chloride channel ClC-1 and analysis of novel mutations leading to Becker-type myotonia. Human Molecular Genetics 3(6): 941-946
29. Lyon, J. and Gorner, P., Altered Fates, 1995, 1st edition, W.W. Norton & Company (New York): p. 349-382

Link to the Neuromuscular Disease Center at the Washington University School of Medicine, St. Louis, MO.