Paramyotonia Congenita and Hyperkalernic Periodic Paralysis are hnked to the Adult Muscle So&um Channel Gene G . C. Ebers, MD," A. L. George, MD,$ R. L. Barchi, MD, PhD,$ S. S. Ting-Passador, MS," R. G. Kallen, MD, PhD,$ G. M. Lathrop, PhD,§ J. S. Beckmann, PhD,9 A. F. Hahn, MD,? W. F. Brown, MD," R. D. Campbell, RN," and A. J. Hudson, MD"

The hyperkalemic periodic paralyses are a clinically heterogeneous group of autosomal dominant syndromes characterized by episodic paralysis associated with an elevated serum potassium level. Affected individuals in the same family tend to have homogeneous symptom complexes, although phenotypic variation is present among different families. For example, myotonia is absent in some pedigrees, present in others, and, in a third variant, paramyotonia congenita, myotonia coexists with cold-induced paralysis. Electrophysiological studies have demonstrated variant-specific abnormalities in skeletal muscle membrane sodium conductance, We tested the hypothesis that hyperkalemic periodic paralysis (without myotonia) and paramyotonia congenita are tightly linked to the tetrodotoxin-sensitive adult skeletal muscle sodium channel gene on chromosome 17q23-25 in two large pedigrees. The DNA polymorphisms detected in the growth hormone skeletal muscle sodium channel complex (GH1-SCN4A) and by flanking polymorphic markers (D17S74 and D17.340) demonstrated no recombinants between the disease phenotypes and this complex. Phenotypic variation in the hereditary hyperkalemic periodic paralyses may result from allelic heterogeneity at the tetrodotoxinsensitive adult skeletal muscle sodium channel locus. Ebers GC, George AL, Barchi RL, Ting-Passador SS, Kallen RG, Lathrop GM, Beckrnann JS, Hahn AF, Brown WF, Campbell RD, Hudson AJ. Paramyotonia congenita and hyperkalernic periodic paralysis are linked to the adult muscle sodium channel gene. Ann Neurol 1991;30:810-816 --

_-

__-_-_____.

-.

____.______

The periodic paralyses are a heterogeneous group of autosomal dominant conditions with variable clinical features and electrophysiological defects [1,2). Hereditary forms have been divided into hypokalemic and hyperkalemic types defined by serum potassium levels during attacks. These disorders occur as autosomal dominant traits with a high degree of penetrance. The status of a normokalemic type is uncertain. There are four identified syndromes of periodic paralysis associated with hyperkalemia: (1) hyperkalemic periodic paralysis (HrKPP), (2) myotonic hyperkalemic periodic paralysis (mHrKPP), (3) paramyotonia congenita (PC), and (4) paralysis periodica pararnyotonica (PPP) 113. Onset of these conditions typically occurs in the first and second decade of life with features of muscular stiffness or weakness lasting minutes to days. In HrKPP, paralysis occurs during the period of rest following exercise and may affect only the exercised muscles. Paralysis may be precipitated by ingestion of potassium-containing foods. Myotonia may be present

or absent. In PC, both myotonia and periodic weakness may be induced by exposure to cold. The relationship of PC to HrKPP has been uncertain. Paralysis or stiffness or both in PC, as in HrKPP in its various forms, may be associated with elevation of serum potassium levels. Attacks of weakness in either condition are relieved by ingestion of carbohydrates and by exercise. Symptoms of myotonia in PC are relieved by administration of lidocaine derivatives, whereas in HrKPP, cold is not ordinarily a provocative factor and paralytic attacks may be prevented by administration of acetazolamide and hydrochlorothiazide { 3}. A progressive vacuolar myopathy may supervene after several years in the periodic paralyses and can be disabling [2). Within families, these conditions generally manifest consistently discrete phenotypes [4). Individuals having either PC or HrKPP phenotypes, however, have been occasionally reported within the same family 151. Although HrKPP can be divided into myotonic, nonmyotonic, and PC variants, a fourth type combines the

From T h e Richard Ivey Centre for Molecular Biology, University Hospital, and ?Victoria Hospital Corporation, London, Ontario, Sciences, Canada; the $David Mahoney Instimte of Ne-logica Universiry of Pennsylvania School of Medicine, Philadelphia, PA; and the §Centre DEtude Polymorphisme Humain, Paris, France.

Received Aug 16, 1991, and in revised form Aug 28. Accepted for publication Aug 29, 1991. Address correspondence to D~Ebers, university ~ ~ Canada N6A 5A5. dermere Rd, London,

810 Copyright 0 1991 by the American Neurological Association

~3 39 win~ i

~

a

,

m 2.1

m P 12.4

),I

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cold-induced paralytic attacks seen in PC with features of HrKPP and has been termed paramyotonic pmiodic paralysis [b, 71. A key development in the understanding of these disorders was the recognition that attacks in both PC and HrKPP are characterized by abnormal increases in sodium conductance and by depolarization block and paralysis if the muscle fibers were sufficiently depolarited {3, 6, 8, 91. Although tetrodotoxin (TTX), a sodium channel blocker, quickly reversed the excessive depolarization in HrKPP, it was ineffective in restoring membrane excitability to previously depolarized muscle fibers in PC. Thus, the electrophysiological defect differs in these conditions. Nevertheless, on the basis of these findings [8], a defect in a skeletal muscle SOdium channel has been suggested. Recently, some of the molecular characteristics of ion channels, including the voltage-sensitive sodium channel group, have been elucidated [lo-121. Two sodium channels are expressed in skeletal muscle and are differentiated by ontogenic expression and sensitivity to TIX {13, 141. The TTX-insensitive channel is expressed in fetal and denervated muscle {14]. In contrast, the adult muscle sodium channel is TTXsensitive, and partial cloning of complementary DNA for the alpha subunit was recently achieved 1151. The gene has been localized to 17q23.1-25.3 {16], and polymorphisms within it have been identified {l?,181. Fontaine and associates [l7] recently described tight linkage of the myotonic variant of HrKPP to the adult skeletal muscle alpha subunit using one of these polymorphisms. We demonstrate here in two large pedigrees that PC and HrKPP without myotonia are also tightly linked to the same locus. These findings support the view that HrKPP both with and without myotonia as well as PC are closely related conditions and further suggest that the clinical phenotype in these disorders

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may arise from different defects in the same adult muscle sodium channel gene.

Methods Pedigrees The pedigree consists of 65 identified individuals spanning five generations (Fig 1). Twenty had or have HrKPP, of whom 12 were alive and affected and 47 alive and unaffected (including spouses). Patients described attacks as occurring during rest following exercise o r after eating potassium-containing foods. Myotonia was not present on clinical examination. The diagnosis was made by history and physical examination in all affected cases and was confirmed in several members by a prolonged exercise test { 3 ]or by the induction of paralytic attacks using orally administered potassium chloride (KCl), or both [ 19, 201. An elevation of serum potassium levels was observed during both induced and spontaneous paralytic attacks. HYPERKALEMIC PERIODIC PARALYSIS (HTKPP).

MYOTONIC HYPFRKALEMIC PERIODIC PARALYSIS

(mHrKPP).

A family of 17 individuals, of which 6 had mHrKPP, was also studied. Myotonia was observed on clinical examination in all affected family members (pedigree not shown). PAKAMYOTONIA CONGENITA (PC). The PC pedigree has been previously reported [21], and the number of members has since expanded to a total of 69 subjects spanning five generations, of whom 25 were affected with PC (Fig 2). Forty-four were alive and unaffected (including spouses). All PC-affected subjects were personally interviewed and examined (by A. J. H. and G. C. E.) They described cold-induced stiffness and often subsequent transient weakness. PC was confirmed in several individuals by electromyography, with demonstration of cold-induced myotonia and membrane depolarization E22).

Electrophysiological studies were carried out in 3 individuals with HrKPP and 3 with mHrKPP. Myotonia was recorded by needle electromyography in mHrKPP and was found to be absent in HrKPP under normal conditions, which confirmed the clincial assessment. Furthermore, in 2 individuals with HrKPP, paralytic attacks were induced by the oral administration of 10 gm of KCI ELECTROPHYSIOLOGICAL STUDIES.

Ebers et al: Paramyotonia Congenita

811

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Fig 2. Pedigree with paramyotonia congenrta (PC).Shaded symbols indicate affected individuals; or& of alleles for haplotypes ic as listed in Table 1.

[2). Myotonic discharges were not observed in either patient in the resting, paralytic, or recovery phase. These studies were carried out because it has been suggested that electromyography should be performed before concluding myotonia is absent in any individual family {23}. The 59 living members of the HrKF'P family, the 11 living members of the mHrKPP family, and the 52 living members of the PC family underwent venipuncture. Some individuals from the HrKPP and PC families were bled even though they were not informative for linkage. These additional individuals were included in multipoint analyses for ordering loci. DNA was isolated from Ficoll-Hypaque-separated lymphocytes using standard procedures 1241. Samples were obtained from all living and informative members from each of these families. LYMPHOCYTE ISOLATION AND DNA PREPARATION.

Following specific restriction endonuclease digestion, Southern blots of electrophoretically separated genomic DNA were hybridized with DNA probes detecting polymorphisms at the following loci: GH1, SCN4A (probes C6b/hNa2), and D17S40 117, 18, 25, 261. These probes detected biallelic polymorphisms with heterozygosity ranging from 0.33 to 0.5. Two probes (Cbb, hNa2) that detect polymorphisms within the adult skeletal muscle sodium channel gene (locus SCN4A) identified a common 25 kb allele (allele 1) but differed in the size of their respective second alleles. Both SCN4A probes have been derived from defined regions of the skeletal muscle sodium channel complementary DNA sequence and appear to lie no further than 20 kb from one another on 17q (unpublished observations). Genotypes for both C6b and hNa2 were identical in all individuals examDNA PROBES.

812 Annals of Neurology

ined. Endonucleases, chromosomal assignments, allelic frequencies, and allelic designations for these probes are given in Table 1. In addition, the polymorphic variable number of tandem repeats (VNTR) probe D17S74 (heterozygosity, 0.91) was used [27]. Paternity and maternity were proved using the DNA fingerprinting probes 33.6 and 33.15 developed by Jeffreys [ZS}. Data from three pedigrees were analyzed using the computer program LINKAGE. Multipoint mapping was carried out with the identified polymorphisms for the following loci: D17S74, GH1, SCN4A, and D17S40. The highly polymorphic D17S74 was recorded as a tetraallelic locus to permit analysis. Two-point lod scores were obtained for 20 recombination fractions ranging from 0.0 to 0.4. Multipoint analyses were performed using LINKMAP from LINKAGE r29, 301. LINKAGE ANALYSIS.

Results Clinical Observations T h e disease trait was fully penetrant in all three families. In H r K P P 19 of 43 (44%) (see Fig l),in m H r K P P 6 of 11 ( 5 5 % ) , and in PC 24 of 43 (56%) (see Fig 2) at-risk individuals were affected (total, 49 of 97) ( 5 1%). No individual was found to have been asymptomatic when the presence of the disease haplotype was determined. With a single exception, onset of symptoms occurred in the first two decades in all affected individuals from the three families. O n e woman from the H r K P P family developed ambiguous symptoms at age 29; however, provocative testing (administration of oral KCl) produced marked transient paralysis with bulbar and respiratory involvement. This finding confirmed the prediction of haplotype assign-

Vol 30 N o 6 December 1991

Table 1 . Markers Used (Chromosome 1 7q Pn(ymorphisms) Map Location

Locus Symbol

Probe

Enzyme

Allele Frequency

Fragment Size

Allele Designation

Reference

17q 17q22-24

D17S74 GH 1

pCMM86 pGH800

Hinjl HzncII

...

...

25 26

SCN4A

Cbb

BgnI

6.7 Kb 4.5 Kb 25 Kb 7.5 Kb 25 Kb 21 Kb

1 2

17q23.1-2 5.3

VNTR 0.68 0.32 0.26 0.74

...

SCN4A

hNa2

BgAI

0.27

0.73 VNTR

=

1

18

2 1 2

17

variable number of randem repeats.

ments derived from the D N A polymorphisms segregating with the trait in her family. In the same family, one 15-year-old male was said to be affected by his affected father but did not bear the disease haplotype. Provocative testing revealed no clinical electrophysiological abnormalities. One additional individual with mHrKPP was diagnosed as having myasthenia gravis as well as the disease haplotype and was found to have mHrKPP. One case each of nonpaternity and nonmaternity identified by D N A fingerprinting were resolved by appropriate historical confirmation. lndividuals with late-life progression myopathy were identified in both famdies.

Table 2 lists the two-point lod scores for the loci D17S74, G H l , SCN4A, and D17S40 for recombination fractions of theta values 0.00, 0.01, 0.1, and 0.2. Maximum two-point lod scores of 5.97 for HrKPP and D17S74,4.09 for HrKPP and CGb/hNa2,3.79 for PC and GH1, and 0.90 for mHrKPP and GH1 (not given in table) were obtained all at a theta value of 0.00. These data reflect that no recombinants between any HrKPP variant phenotypes and the tightly linked polymorphisms detected by probes GH1, Cbb, and hNa2 were identified in any of the three families. Multipoint mapping did not resolve the relative order of GH1, CGb, and hNa2. Recombinants for Lew 101 (D17S40) place this marker telomeric to this complex (data not shown). D17S74 and D17S40 are polymorphic markers that flank the GH1-SCN4A-HrKPP complex and are centromeric and telomeric at a distance of 2.5 and 12.5 cM, respectively (Beckman JS, Phillips J. Unpublished observations, 1991).

DNA Polymorphisms The disease haplotype in the HrKPP family for the tightly linked complex of GH1-SCN4A (pGH800C6b-hNa2) was (1,2,2), for the mHrKPP family (2,2,2), and for the PC family ( l , l , l ) (see Table 1 for allelic designations). These findings indicate that each of the three families bears a different disease haplotype. The mHrKPP family, however, bears the same alleles for G H 1 and C6b-hNd, the only two markers tested by Fontaine and colleagues [I71 in their linkage study of *what appears to be the same variantmHrKF'P.

Discussion The hereditary forms of periodic paralysis have been classified clinically by changes in serum potassium levels during paralytic attacks. Although the status of the normokalemic variety is uncertain, hypokalemic and hyperkalemic periodic paralyses represent unambigu-

Table 2. Two-Point Lod Scores (MLINK)"

Hyperkalemic Periodic Paralysis

Locus

Paramyotonia Congenita

Recombination Fraction Theta Value (0)

Dt7S74 GH 1

SCN4A D 17S40

0.00

0.01

0.1

0.2

0.00

0.01

0.1

0.02

5.97b 1.58 4.09b 1.42

5.89 1.53 4.01 1.42

5.00 1.14 3.34

3.72 0.69 2.53 0.87

inF

2.40 3.74 0.38 1.34

3.45b 3.21 0.35

2.91 2.46 0.28 0.90

1.23

3.79b 0.38 1.36

1.16

"MLINK technique from f297. bMaxirnum score. 'Not informative,

inf = infinite.

Ebers et al: Paramyotonia Congenita

813

Table 3. The Hypwkalemic Pwiodic Pavalyjis

Feature {Refs}

HrKPP

mHrKPP

PC

Inheritance [3,6,8,213 Putative gene defect 117,181

Dominant ASkMNa+ channel (17q 23-25) 1st decade Infrequent

Dominant ASkMNa + channel (17q 23-25) 1st decade Infrequent

Dominant Dominant ASkMNa + channel > (17q 23-25) 1st decade 1st decade Probably infrequent Rare

No

Yes Yes

Yes

Yes

No

Yes

t

+

Yes May be absent

+

Rest after exercise, KC1 Mild exercise, glucose, acetazolamide

Rest after exercise, cold, KCl Mild exercise, glucose, thiazides, acetazolamide

Age of onset [3,6,2 11 Myopathy {21)

PPP

Myotonia {2,6,2 l}

Mechanical Electrical Potassium sensitivity { 1,6,211 'ITX depolarization block {7,8} Provocative factors [1,2,6,8,9,21) Palliative factors C1,2,6,8,91

+

+

Cold, exercise, often KCI Glucose, tocainide, acetazolamide, mild exercise

+

7

Rest after exercise, cold, KCI Mild exercise, thiazides, tocainide

HrKPP = hyperkalemic periodic paralysis without myotonia; mHrKPP = hyperkalemic periodic paralysis with myotonia; PC congenita; PPP = paramyotonia periodic paralysis; ASkM = adult skeletal muscle; KC1 = potassium chloride.

ously distinct entities. Nosology within the hyperkalemic group, however, has been uncertain. The clinical syndromes within individual families have generally been consistent in terms of clinical pattern and electrophysiological findings [2]; however, clinical and electrophysiological features suggest that there are at least four distinct variants of hyperkalemic periodic paralysis: HrKPP, mHrKPP, PC, and PPP. Some genetic clinical and electrophysiological features are summarized in Table 3. The relationship among these disorders has been unclear; however, the existence of these variants as discrete entities has been supported by several electrophysiological observations. These findings include the variable presence of myotonia from family to family, the lack of sensitivity to potassium loading in some PC families, the worsening of myotonia with repetitive muscle contraction in PC, electrical silence in coldinduced paralysis in PC, and a variety of membrane defects inferred from in vitro electrophysiological studies [12, 22, 31-33]. Lehmann-Horn and colieagues [ 8 ] showed that muscle fibers in mHrKPP were spontaneously active in both normal and high potassium solutions and that a close correlation existed between paralysis and the degree of membrane depolarization. In HrKPP, muscle fibers were not spontaneously active and paralysis occurred in a high potassium solution at which normal muscle was still excitable. In PC, the distinguishing feature from either HrKPP or mHrKPP was the coldinduced muscle fiber inexcitability. Moreover, weakness in PC responds to administration of tocainide, whereas HrKPP and mHrKPP respond only to administration of thiazide diuretics and beta-adrenergic drugs. Despite these distinctions there are the families

814 Annals of Neurology Vol 30

=

paramyotonia

with PPP that resembles both HrKPP and PC [6]. Studies of muscle fiber membrane potentials and ion permeability have shown that despite clinical and electrophysiological differences, the hyperkalemic group of disorders appears to share a common defect consisting of abnormal sodium conductance with an excessive accumulation of sodium within muscle fibers. Nevertheless, despite these informative investigations, it has remained unclear whether heterogeneity is the result of defects at different loci or is due to allelic heterogeneity. We have shown tight linkage of two forms of hyperkalemic periodic paralysis (HrKPP and PC) to the SCN4A locus. For HrKPP, highly significant two-point lod scores of 5.97 for D17S74 and 4.09 for C6b were obtained. The higher score for D17S74 reflects the greater informativeness of this polymorphic marker even though recombinants place this locus 7 cM telomeric to the GH1-SCN4A complex. Similarly, for PC, the maximum lod score of 3.79 for GH1 at a theta value of 0.00 is only slightly greater than the 3.45 obtained for D17S74 at a theta value of 0.01. Although not statistically significant, the absence of recombinants in our smaller family with mHrKPP is consistent with the recent report linking this variant with the same locus [17]. These findings are consistent with electrophysiological studies previously carried out in this group of disorders that suggested an abnormality in the voltage-gated sodium channel. The studies reported here support the view that clinical or electrophysiological heterogeneity, or both, among the periodic paralyses associated with hyperkalemia are due to allelic heterogeneity. The haplotypes for alleles at GH1, C6b, and hNa2 were different in the three variants reported in this study, con-

No 6 December 1991

sistent with this notion. Furthermore, the mHrKPP variant reported here bears the same haplotype as in the mHrKPP family reported by Fontaine and associates [171, in which linkage was first demonstrated. This is consistent with a common ancestor for both these families or linkage disequilibrium. Typing of further families will be required to see if variant-specific haplotypes are present. The success of the candidate gene aproach in identifying linkage in this group of conditions appears to unify this group of disorders and strongly implicates the voltage-gated sodium channel as the location of the fundamental genetic defect. It would seem probable that the heterogeneous features of this group of conditions may result from defects in the different locations within the coding or adjacent regulatory sequences of the TT.X-sensitive skeletal muscle sodium channel gene. At least for the three variants now linked to the adult skeletal muscle sodium channel gene, the use of these probes and identification of diseasebearing haplotypes could be useful for the diagnosis of ambiguous cases and for presymptomatic diagnosis in the offspring of affected individuals from informative pedigrees. Although these linkage studies are very strongly suggestive, additional studies aimed at identifying the precise molecular defects in these disorders will be required to formally prove that the defect responsible for these conditions lies within the adult muscle sodium channel gene itself. This identification may allow correlation of the molecular pathology with the disease phenotype and enhance our understanding of the structure and function of the skeletal muscle sodium channel. Addendum Evidence linking paramyotonia congenita to the adult muscle sodium channel has also been reported by L. J. Ptacek and co-workers in an article currently in press for the American Journal of Human Genetics. This study was supported by the Medical Research Council of Canada (MRI-MT-10471; A. J. H., G. C. E.); National Institutes of Health grants NS-18013 and NS-08075; the PEW foundation; the Veterans Administration; and the Muscular Dystrophy Association of America. We wish to thank K. Davies, G. Rouleay J. Roemmens. R. Dunn, D. Bulman, and D. W. Cox for helpful discussions. These studies were initiated while one of the authors (G. C. E.) was a visiting scholar at Green College and the Nuffield Department of Medicine, Oxford. We wish to thank Eric Hoffman for the hNa2 probe, Guy Rouleau for LEW101, and A. Jeffreys for 33.6 and 33.15. We also wish to thank the patients and their relatives for uniform cooperation as well as Cathy Marsh for typing the manuscript.

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816 Annals of Neurology Vol 30 No 6 December 1991

clonality in gastrointestinal cancer by D N A fingerprinting.J Clin Invest 1988;82: 1532- 1537 Lathrop GM, Lalouel JM, Julier C, Ott J. Strategies for multilocus linkage analysis in humans. PNAS 1984;81:3443-3446 Lathrop GM, Moue1 JM, Julier C, Ott J. Multilocus linkage analysis in humans; detection of linkage and estimation of recombination. Am J Hum Genet 1985;37:482-498 Haass A, Ricker K, Rudel R, et al. Clinical study of paramyotonia congenita with and without myotonia in a warm environment. Muscle Nerve 1981;4:388 Lehmann-Horn F, Rudel R, Dengier R, et al. Membrane defects in pai-amyotonia congenita with and without myotonia in a warm environment. Muscle Nerve 1981;4:396-406 Rudel R, Lehmann-Horn H. Membrane changes in cells from myotonia patients. Physiol Res 1085;65:310-356