Neuromusc. Disord., Vol. 3. No. 2, pp. 161-168, 1993 0960-8966/93 $6.00 * 0.00 Printed in Great Britain Pergamon Press Ltd

WORKSHOP REPORT*

NON-DYSTROPHIC MYOTONIAS AND PERIODIC PARALYSES A European Neuromuscular Center Workshop held 4-6 October 1992, Ulm, Germany

INTRODUCTION Muscle diseases: congenita (Thomsen) (MIM Our understanding of the pathology of the non- 160800); dystrophic myotonias and the periodic para- Recessive generalized myotonia (Becker) lyses has profited immensely from the use of (MIM 255700). modern electrophysiology (three microelectrode voltage clamp, patch-clamp techniques) and The pathomechanism of the very rare molecular biology (candidate gene approaches Schwartz-Jampel syndrome (SJS) has not yet in contrast to reverse genetics in other neuro- been sufficiently clarified. Classification is muscular diseases). In the past few years it has therefore not possible and the syndrome was become clear that--apart from the not yet not discussed much during the workshop. There understood pathomechanism of myotonia in are, however, plans for a future ENMC work- --there are two clearly shop on Schwartz-Jampel Syndrome and the distinct pathomechanisms discernible for the authors of this report call for signs of interest! non-dystrophic myotonic disorders: with a In hypokalemic , the most mutation in either the gene encoding the common form of the periodic paralyses (which C1- channel (CHLCNI) or the is never associated with myotonia), the mutated gene encoding the ~-subunit of the adult skel- gene, has not yet been localized. Electrophysio- etal muscle Na ÷ 'channel (SCN4A). Several logical evidence suggests that a K + channel mutations exist in each gene. might be altered in this disease, and there is a As a consequence of this new knowledge the recent report where the authors excluded terms of skeletal muscle CI- channel diseases linkage to SCN4A [3]. For these reasons the (chloride ) and skeletal muscle disease is different from the mainstream of the Na ÷ channel diseases (sodium channelopathies) workshop and was not a matter of discussion. were coined [1, 2] and the workshop (organized The participants agreed unanimously that it by Frank Lehmann-Horn and Reinhardt was desirable to use clear abbreviations for the Rfidel) was structured accordingly. The different forms of periodic paralysis and decided participants of the workshop approved of this on HyperPP, NormoPP and HypoPP for classification and suggested that the Editor of hyperkalemic, normokalemic and hypokalemic Neuromuscular Disorders be asked to adopt it periodic paralysis, respectively. Paramyotonia for his Table of Gene Locations. congenita is often abbreviated to PC. Professor Emery suggested that the partic- Muscle diseases: ipants form an international consortium with Hyperkalemic periodic paralysis (MIM Professor Riidel as chairman. It was agreed that 170500); it was desirable to convene again after about 2 Normokalemic periodic paralysis (MIM yr. 170600); Paramyotonia congenita (MIM 168300); Myotonia fluctuans. MUSCLE SODIUM CHANNEL DISEASES

For many years clinicians have argued as to whether the two major members of this group, *The authors dedicate this report to Professor Peter Emil Becker who will celebrate his 85th birthday in the autumn paramyotonia congenita and hyperkalemic of 1993. periodic paralysis, are separate diseases or

161 162 Workshop Reporl

comprise a nosological entity. This problem can Na' current (Stephen Cannon [9]: [,ouis be regarded as solved, as it is now clear that Ptfi(zek). Walter Stfihmer pointed out that the various mutations of the SCN4A gene exist and $5/$6 segment region in each channel repeat that each mutation causes a typical clinical may be involved in channel inactivation. He picture. The term "adynamia--paramyotonia then took one of the shaker related K* complex", coined by King Engei, making use of channels as an example to discuss how much the original name given by Gamstorp to the channel open probability can be reduced by HyperPP, thus seems to be a fortunate clinical low extracellular K *. designation for the muscle Na* channel The second session, chaired by Thomas Deufel, diseases. Two sessions and a general discussion dealt with the genotypes, electrophysiology, were devoted to this subject. The first session and phenotypes of muscle Na- channel with Walter Stiihmer in the chair, dealt with diseases. Several groups presented new SCN4A genomic organization, gene expression and mutations and described their relation to vari- protein structure of the human skeletal muscle ous clinical pictures. Four of the mutations Na + channel. Two groups (Andrea McClatchey, were related to paramyotonia congenita or Al George) reported on their completion of the atypical myotonia (Frank Lehmann-Horn, identification of the exon/intron structure of the Andrea McClatchey, Louis Pt/leek), another human SCN4A gene encoding the ~z-subunit of was associated with paramyotonia congenita the adult skeletal muscle Na + channel. The with cold- and potassium-induced stiffness gene contains 24 exons distributed over about (Roland Heine), two further ones were related 30 kb of chromosome 17q23. As with many to hyperkalemic periodic paralysis (Frank genes, the genomic structure becomes more Lehmann-Horn) and the last was linked to a condensed towards the 3' end, with the last family with the symptoms of hyperkalemic 30% of the coding sequence appearing in a periodic paralysis and cold-induced weakness. single exon [4, 5]. This family shows incomplete penetrance Data on other Na + channel genes (different (Andrea McClatchey). These new mutations mammalian species and different human tissues) bring the number of known human SCN4A were presented for comparison by A1 George mutations to 13, all causing either cold-induced and John Caldwell. About ten different, but stiffness or potassium-induced paralysis or closely related, Na + channel ~z-subunit genes atypical myotonias. All mutations result in the are known, most of which are expressed in change of a single amino acid in the Na + brain, peripheral nerves and muscle [4, 6, 7]. While channel protein (Table 1) altering its function. hSkM1, the TTX-sensitive SCN4A product, is and are transmitted as dominant traits. Many only expressed in adult human skeletal muscle, of the amino acid changes are located either another two distantly related ~-subunits, both within the intracellular loop connecting repeats with low TTX sensitivity, were found in fetal III and IV, the supposed inactivation gate of and denervated skeletal muscle and in adult the channel, or at the intracellular side of the cardiac muscle (hill), as well as in myometrium $5/$6 interlinker, a region hypothesized to act and fetal skeletal muscle (hH2) [8]. No diseases as an acceptor of the inactivation gate (Fig. 1). have so far been linked to any Na + channel Patch clamping of native muscle preparations gene other than to SCN4A, but it was suggested from patients with hyperkalemic periodic par- that there might be such diseases. alysis or paramyotonia congenita, revealed an The expression of the human Na + channel ~- increase in the time constant of fast Na ~ subunit or its equivalent in the rat, rSkMl, in channel inactivation (Frank Lehmann-Horn). Xenopus oocytes has been successful but the The number of late Na ÷ channel openings was expression system was not satisfactory because also much higher than the controls [13. 20]. of an abnormally slow inactivation of the normal Resealed fiber segments showed long-lasting channel (A1 George). When hSkM1 was trans- trains of action potentials which came to an end fected into mammalian cells, e.g. human at a reduced resting potential. In muscles from embryonic kidney cells (HEK), inactivation was PC patients the reduced resting potential was at about normal. Introduction of the equivalent of about - 60 mV, with the fibers still excitable, or the C2111T and A4774G mutations into the rat at -40 mV, at which point the fibers were construct and its expression in HEK cells inexcitable. In muscles from HyperPP patients, resulted in an altered mode of gating with the runs usually ended around -40 mV. Com- repetitive re-openings producing a persistent puter simulation of action potentials Workshop Report 163

Table 1. Genotype/phenotype correlation in patients with a muscle Na + channel disease (lines 1-13), and silent amino acid substitutions found in healthy controls (lines 14--19). Note that a Phe 1421 Leu substitution was found for Quarter horses [10]

Genotype CHannel Predicted Exon Phenotype Reference part substitution

C2188T* I1S5~ Thr704Met 13 HyperPP I 1 C2411T IIS6~ Ser804Phe 14 PC 5 G3466A (IIIS4/5)~ Alal 156Thr 19 HyperPP 5 G3917T (III/IV)~ Glyl306Val 22 PC 12 G3917A (III/IV), Glyl306Glu 22 Myotonia permanens 13 G3917C (III/IV)~ Glyl306Ala 22 Myotonia fluctuans 13 C3938T (III/IV)~ Thrl313Met 22 PC 12 A4078G IVS 1 Met 1360Va1 23 HyperPP t T4298G IVS3 Leu 1433Arg 24 PC 14 C4342T IVS4 Arg1448Cys 24 PC 15 C4343A IVS4 Arg1448His 24 PC 15 G4765A IVS6~ Val1589Met 24 PC 16 A4774G IVS6~ Met1592Val 24 HyperPP 17 607A/G 1S3 203Met/Val 4 Normal 4 2578G/A (ll/llI)~ 860Glu/Lys 14 Normal 4 2580G/T (II/III)~ 860Glu/Asp 14 Normal 18 2794G/C (II/III)~ 932Asp/His 14 Normal 14 4126G/A IVSI/S2 c 1376Asp/Asn 23 Normal 16 4817A/G IVS6~ 1606Glu/Gly 24 Normal 19

l-IV = number of repeats in the Na + channel protein. SI-S6 = number of the transmembrane segment within each repeat. i = intracellular loop or "inactivation-gate acceptor" at the intracellular pore lining. e = extracellular loop. * = corresponding to C2111T in [11]; 2188 is correct in relation to other numbers. t = reported by Lehmann-Horn for a HyperPP family with cold-induced weakness without stiffness,

epea, Outside

Inside

coo-

Fig. 1. Diagram of the sodium channel protein showing the locations of the amino acids that are replaced in patients with diseases of the adynamia-paramyotonia complex. demonstrated that the failure of inactivation of phenotype correlations (Louis Pt~i~ek, Eric a small proportion (less than 2%) of Na + Hoffman, Frank Lehmann-Horn). Each mutation channels can cause repetitive activity ending in causes either HyperPP or PC. Thr704Met seems a variable degree of stable depolarization (Stephen to be the most frequent amino acid substitution Cannon). Accumulation of K + within the T (6 out of 17 German HyperPP families). This tubules was a necessary feature of the computer mutation results in a HyperPP subtype char- model for repetitive activity to occur [21, 22]. acterized by drug resistance and permanent A novel dominant mutation in the homo- weakness of mainly late onset. The same muta- logous horse gene was shown to be the tion was found in a family diagnosed as having molecular basis of periodic paralysis in Quarter normokalemic periodic paralysis on the basis of horses [10]. This horse disease was inadvertently the original clinical criteria [23]. Furthermore, a disseminated by breeders, as the mutation causes clinically convincing potassium-induced para- the desirable muscle hypertrophy. Up to 5% of lysis family from Yugoslavia was presented this popular breed (3,000,000 currently registered which did not show linkage to the SCN4A gene: in the U.S.A.) are affected (Eric Hoffman). this was the first example of genetic hetero- The discussion focused on additional linkage geneity within hyperkalemic periodic paralysis studies (Bertrand Fontaine) and on genotype/ (Eric Hoffman). In addition, dinucleotide 164 Workshop Report

haplotype/mutation correlations (Andrea membrane-spanning domains, in accordance McClatchey, Eric Hoffman) and normal poly- with the sizable lack of C1- conductance [27]. morphisms were discussed (see Table 1). In two other mouse mutants the Chlcnl genes are inactivated by point mutations (Gronemeier M, Prosser J, Steinmeyer K, Jentsch Th. MUSCLE CHLORIDE CHANNEL DISEASES Jockusch H. Nonsense and missense mutations in the muscular C'I channel gene Chtcnl of The low chloride conductance theory of myotonic mice. In preparation). myotonia was first proposed in the 1960s by The molecular genetics of human congenital Shirley Bryant, on the basis of electrophysio- myotonia was presented by Manuela Koch. In logical studies on goats with autosomal 1992, a partial cDNA of the human CLC-I dominant . It explains the channel was cloned and physically localized on hyperexcitability as a direct consequence of a chromosome 7q32-ter. Linkage was shown to reduced CI- conductance in the T system of the TCRB locus supporting the mouse-human myotonic fibers. Later voltage clamp studies homology map for this chromosomal region showed that this theory applies to myotonia in [24]. Tight linkage between Thomsen myotonia all Becker myotonia patients. Surprisingly, in and the TCRB locus was shown by a Canadian several of the patients characterized by dom- group [28] and, at the same time, linkage to this inant inheritance, the C1- component locus was found for German Thomsen and conductance was normal. In addition, Becker families [24]. An unusual restriction site abnormalities in the properties of the Na ÷ in the CHLCN1 genes of two Becker myotonia channels were noted in muscles from both families revealed a T-to-G transversion Thomsen and Becker patients. It was not until predicting a phe-to-cys substitution in the 8th 1992 that linkage to the gene coding for the of the putative 12 transmembrane domains. muscle C l- channel was proven for both The molecular genetic data suggest that Becker and Thomsen myotonia [24]. different mutations in the CHLCN1 gene may The first of two sessions on the "muscle cause dominant or recessive myotonia. chloride channel diseases", chaired by Manuela The second session on muscle C I channel Koch, was devoted to the molecular biology of diseases was chaired by Erich Kuhn, with elec- the muscle CI- channel, the CHLCN1 gene trophysiology as the main subject. In spite of product. Muscle C1- channels are members of many attempts in several laboratories around a family of voltage-gated C l channels that are the world, the C1 single-channel activity has completely different from the C I channels not been consistently recorded from native Which are altered in [25]. They muscle preparations, although it can be easily provide 4/5 of the CI conductance of the measured in myoballs cultured from normal muscle fiber membrane. Klaus Steinmeyer individuals and myotonia patients. Christoph reported that the CHLCN1 protein consists of Fahlke reported that the single-channel con- 991 amino acids and that its molecular weight is ductance of the most frequent ("intermediate") 110 kDa. The mRNA contains 4-5 kb. In the C1- channel in myoballs was reduced to 50% rat, its expression greatly increases within the for patients with Becker myotonia [29]. While first 30 days of postnatal development. this myoball channel has not yet been The report was preceded by a short review on conclusively shown to be expressed in adult the myotonic ADR mouse by Harald Jockusch. skeletal muscle, Erhard Wischmeyer presented Similar to the role the myotonic goat played for data on CI- channels found in lipid-supple- the elucidation of the electrophysiological basis mented vesicles prepared from the sarcolemmal of C 1- channel myotonia, the mutation adr and fraction of adult skeletal muscle of rabbit [30] some other allelic mutations played an and mouse. An indanyloxyacetic acid-sensitive important role in the discovery of human and partially rectifying CI- channel was only recessive myotonia as a C1- channel disease. present in the wild-type but not in the ADR The adr locus, and later the Chlcnl gene, were mouse, and is, therefore, a candidate for the shown to be linked to the T cell receptor [3 product of the Chlcnl gene. (Tcrb) and the Hox loci on chromosome 6 In the final portion of the workshop, [26, 27]. A transposon of the ETn family was secondary changes in the gating of Na ÷ found to be inserted into the adr allele that channels in "muscle chloride channel diseases" destroys its coding potential for several were discussed by Paul Iaizzo. The problem of Workshop Report 165 the patients with dominant myotonia and INTERNATIONAL ABBREVIATIONS USED normal CI- conductance [31] was solved in part when it was recognized that myotonia SCN4A Gene encoding the human adult fluctuans is a Na- channel disease. There is, skeletal muscle Na ÷ channel; however, still the unexplained problem that hSkM 1 human adult skeletal muscle Na ÷ some Thomsen patients had normal C1- channel (product of the SCN4A conductance and slowed inactivation of the gene); Na ÷ channels, rSkM 1 rat adult skeletal muscle Na ÷ The electrophysiological data were also channel; contrasted to those observed for Schwartz- hill gene encoding the human cardiac, Jampel syndrome as "food for thought". fetal and denervated skeletal The workshop closed with a discussion, muscle Na ÷ channels; led by Reinhardt Rfidel, on the multimeric hH2 gene encoding the human structure of the C1- channel and the pos- myometrium and fetal skeletal sible disturbance of function caused by muscle Na ÷ channels; the mutations. As a last point, the secondary adr adr gene; abnormalities in the C1- channel diseases ADR phenotype of the mouse mutant were brought up. The abnormal K ÷ and Na ÷ carrying two adr alleles; conductances already reported by Bryant Chlcn 1 mouse muscle C1- channel gene; for the myotonic goat are unexplained, as CHLCN1 human muscle C1 - channel gene; are the Na ÷ channel abnormalities in Clc- 1 probe and locus for the mouse Becker and Thomsen patients mentioned above. muscle C1- channel gene; In the myotonic ADR mouse, symptomatic CLC-1 probe and locus for the human C1 - treatment with the Na ÷ channel blocker channel gene; tocainide, partially reversed electro- RFLP restriction fragment length poly- physiological and biochemical altera- morphism; tions. Tcrb mouse T cell receptor 13; The participants showed their gratitude to TCRB human T cell receptor 13. the workshop sponsors, the European Neuromuscular Center (ENMC), the Associa- DIAGNOSTIC CRITERIA tion Franqaise Contre les (AFM), the University of Ulm and the Deutsche For- Since the genes and the gene products are schungsgemeinschaft (DFG). known in the principal diseases of non-dys- trophic myotonias and periodic paralyses, and since an increasing number of molecular bio- List of participants logical laboratories have the relevant genetic markers available, an exact diagnosis will, in J. H. Caldwell (Denver), S. Cannon future, be made by the identification of the (Boston), T. Deufel (Mfinster), A. E. Emery mutation. At present, many laboratories are (Edinburgh), C. Fahlke (Ulm), B. Fontaine engaged in correlating the clinical symptoms of (Paris), A. L. George (Nashville), E. P. Hoffman their individual families with the various muta- (Pittsburg), P. A. Iaizzo (Minneapolis), tions and, therefore, a precise statement of the H. Jockusch (Bielefeld), M. Koch (Marburg), clinical diagnostic criteria remains useful. E. Kuhn (Heidelberg), F. Lehmann-Horn (Ulm), It is important to state that myotonia, i.e. A. McClatchey (Boston), L. Pt~rek (Salt Lake muscle stiffness, is a symptom that can be City), K. Ricker (Wiirzburg), R. Riidel (Ulm), present in both muscle C1- and Na ÷ channel P. Steinbach (Ulm), K. Steinmeyer (Hamburg), diseases (and, of course, also in myotonic dys- W. Stfihmer (Grttingen), E. Wischmeyer trophy and Schwartz-Jampel syndrome). The (Bielefeld). myotonia is best assessed as myotonic runs in the electromyogram. Diagnostic differentiation of the various diseases on the mere basis of ENMC these runs is not dependable. Muscle biopsy is usually not helpful for establishing the M. R. Rutgers (Baarn). diagnosis. 166 Workshop Report

The class of C I channel diseases comprises Family history. Autosomal recessive inherit- dominant myotonia congenita (Thomsen) and ance. Some of the heterozygous carriers sho~ recessive generalized myotonia (Becker). The myotonic runs in the EMG. Such cases must term of myotonia congenita should only be not be confused with dominant myotonia, and reserved for these C I - channel diseases. sometimes molecular biology is required to The class of Na ÷ channel diseases encom- differentiate from myotonic dystrophy. passes all clinical variants of the "adynamia Age ~?["onset. Occasionally present in early paramyotonia complex". Although the key childhood, usually first decade of life, in some symptoms, namely attacks of muscle weakness cases not before the end of the second decade and episodes of muscle stiffness, are known to and even progression of symptoms into the overlap to various degrees, it makes sense from third decade of life. a clinical point of view to maintain the Clinical signs. Muscle stiffness, particularly differentiation between hyperkalemic periodic after rest, muscle function improving with con- paralysis (identical with Gamstorp's adynamia tinuing exercise (warm up). In many patients episodica hereditaria) and paramyotonia marked transient weakness after rest which congenita (Eulenburg) because preventive improves during several minutes of continued measures are different for the two symptoms. exercise. Weakness is more pronounced in the HyperPP also implies a possible prognosis of upper extremities, stiffness is more pronounced progressive permanent weakness that is not a in the legs. In many cases hip and leg muscles feature of PC. are hypertrophied. The signs are usually pro- gressive for a few years after their first Dom&ant myotonia congenita appearance and then remain stable for the rest of the life. The usual (but very rare) form is Thomsen's Clinical signs that must not be mistaken. The disease. There is also a form that is dis- well-known phenomenon of anticipation in tinguished by very mild myotonia (DeJong's myotonic dystrophy may lead to a familial myotonia levior). It remains to be discovered constellation suggesting recessive inheritance whether this is caused by an allelic mutation. and, as a consequence, may lead to the spurious diagnosis of recessive generalized myotonia. On Family history. Autosomal dominant inheri- the other hand, misinterpretation of the trans- tance; 100% penetralace. ient weakness may lead to the spurious Age of onset. From birth to early childhood. diagnosis of myotonic dystrophy. In older Clinical signs. Muscle stiffness, particularly patients with recessive generalized myotonia after rest, muscle function improving with con- muscle biopsies may show a morphologic pat- tinuing exercise (warm up). Myotonia fluctuates tern that can be misdiagnosed as muscular only slightly during lifetime; no progression. dystrophy. Frequent muscle hypertrophy. Clinical signs that must not be mistaken. There are cases of Na ÷ channel disease having Paramyotonia congenita myotonia without any weakness. The myotonia The classical form was described by may exist without cooling. Before the advent of Eulenburg and independently by Rich. Several molecular biology, these cases were often mis- mutations in the Na + channel gene result in the diagnosed as forms of myotonia congenita. classical clinical picture. Although patients with myotonia congenita, Family history. Autosomal dominant in- when asked, often state that their stiffness in- heritance; 100% penetrance. creases in the cold, this cannot be substantiated Age of onset. From birth. with objective measurements of muscle relaxa- Clinical signs. Muscle stiffness increasing with tion times. exercise (paradoxical myotonia), In many families paramyotonia is dramatically increased Recessive generalized myotonia when the muscles are exercised in the cold: At least two mutations must exist causing the Transition from stiffness to local weakness same clinical picture. (There is one mutation when muscles are extensively exercised in the detected in several unrelated families, but other cold. Recovery from weakness may last several investigated families do not show this hours. Cave: Some families present consistently mutation.) cold- and exercise-induced stiffness without Workshop Report 167 weakness. These are often misdiagnosed as hyperkalemia is a variant of the adynamia- having myotonia congenita! paramyotonia complex. Variability of signs. There are families where affected members present with the classical symp- Myotonia fluctuans toms of paramyotonia congenita and also often Three families have been studied. Linkage to experience attacks of hyperkalemic paralysis. the Na+channel gene is established. The presentation of both sets of symptoms in Family history. Autosomal dominant severe form was termed paralysis periodica inheritance. paramyotonica (PPP) by P. E. Becker, however, Age of onset. Early childhood. a continuum seems to exist, with PPP families Clinical signs. Severe generalized stiffness with- and families having "pure" paramyotonia out weakness following exercise and/or K ÷ congenita presenting the two extremes. The loading. Rest after exercise leads to "delayed- severity of stiffness is not the same in all para- onset myotonia". Cold does not induce stiffness or myotonia families. weakness. Clinical signs that must not be mistaken. Permanent weakness is not observed in para- FRANK LEHMANN-HORN, myotonia congenita. REINHARDT RODEL and KENNETH RICKER Hyperkalemic periodic paralysis Department Physiologic Several mutations in the Na ÷ channel gene Universit/it Ulm may lead to the classical clinical picture. Ulm Family history. Autosomal dominant Germany inheritance; complete penetrance, but severity is very variable. Age of onset. Early childhood to second REFERENCES decade of life. Clinical signs. Attacks of weakness, usually in 1. Lehmann-Horn F, Engel A G, Ricker K, R/idel R. Periodic paralyses and paramyotonia congenita. In: the morning, lasting from 10 min to 1 h or so, Engel A G, Franzini-Armstrong C, eds. Myology, 2nd very rarely up to 1-2 days. Some patients Edn. New York: McGraw-Hill, in press. experience only a few attacks of weakness in 2. Riidel R, Lehmann-Horn F, Ricker K. 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