A sodium channel defect in hyperkalemic periodic paralysis: potassium-induced failure of inactivation.

Hyperkalemic periodic analysis (HPP) is an autosomal dominant disorder characterized by episodic weakness lasting minutes to days in association with a mild elevation in serum K+. In vitro measurements of whole-cell currents in HPP muscle have demonstrated a persistent, tetrodotoxin-sensitive Na+ current, and we have recently shown by linkage analysis that the Na+ channel alpha subunit gene may contain the HPP mutation. In this study, we have made patch-clamp recordings from cultured HPP myotubes and found a defect in the normal voltage-dependent inactivation of Na+ channels. Moderate elevation of extracellular K+ favors an aberrant gating mode in a small fraction of the channels that is characterized by persistent reopenings and prolonged dwell times in the open state. The Na+ current, through noninactivating channels, may cause the skeletal muscle weakness in HPP by depolarizing the cell, thereby inactivating normal Na+ channels, which are then unable to generate an action potential. Thus the dominant expression of HPP is manifest by inactivation of the wild-type Na+ channel through the influence of the mutant gene product on membrane voltage.     

Paramyotonia congenita and hyperkalemic periodic paralysis map to the same sodium-channel gene locus.

Paramyotonia congenita (PC), an autosomal dominant muscle disease, shares some clinical and electrophysiological similarities with another myotonic muscle disorder, hyperkalemic periodic paralysis (HYPP). However, clinical and electrophysiologic differences allow differentiation of the two disorders. The HYPP locus was recently shown to be linked to a skeletal muscle sodium-channel gene probe. We now report that PC maps to the same locus (LOD score 4.4, theta = 0 at assumed penetrance of .95). These linkage results, coupled with physiological data demonstrating abnormal sodium-channel function in patients with PC, implicate a sodium-channel gene as an important candidate for the site of mutation responsible for PC. Furthermore, this is strong evidence for the hypothesis that PC and HYPP are allelic disorders.     

Evidence for a single pedigree source of the hyperkalemic periodic paralysis susceptibility gene in Quarter Horses

The pedigree origin of a base pair substitution in the horse muscle sodium channel gene that confers susceptibility to the muscle disease hyperkalemic periodic paralysis (HYPP) was investigated with a set of 978 Quarter Horses. The horses were chosen at random, based on a collection of blood samples taken between 1989 and 1991 to meet parentage testing requirements, primarily but not exclusively from breeding stallions. The frequency of Quarter Horses positive for the base pair substitution, all heterozygotes, was 4-4%, which corresponds to an allelic frequency of 0.02. All horses positive for the gene traced to a single previously identified stallion as first, second or third generation descendants. A higher frequency of the HYPP susceptibility trait than expected by random occurrence was found among his descendants in this study.     

Identification of a mutation in the gene causing hyperkalemic periodic paralysis.

DNA from seven unrelated patients with hyperkalemic periodic paralysis (HYPP) was examined for mutations in the adult skeletal muscle sodium channel gene (SCN4A) known to be genetically linked to the disorder. Single-strand conformation polymorphism analysis revealed aberrant bands that were unique to three of these seven patients. All three had prominent fixed muscle weakness, while the remaining four did not. Sequencing the aberrant bands demonstrated the same C to T transition in all three unrelated patients, predicting substitution of a highly conserved threonine residue with a methionine in a membrane-spanning segment of this sodium channel protein. The observation of a distinct mutation that cosegregates with HYPP in two families and appears as a de novo mutation in a third establishes SCN4A as the HYPP gene. Furthermore, this mutation is associated with a form of HYPP in which fixed muscle weakness is seen.     

Dinucleotide repeat polymorphisms at the SCN4A locus suggest allelic heterogeneity of hyperkalemic periodic paralysis and paramyotonia congenita

Two polymorphic dinucleotide repeats--one (dGdA)n and one (dGdT)n--have been identified at the SCN4A locus, encoding the alpha-subunit of the adult skeletal muscle sodium channel. When typed using PCR, the dinucleotide repeats display 4 and 10 alleles, respectively, with a predicted heterozygosity of .81 for the combined haplotype. We have applied these polymorphisms to the investigation of hyperkalemic periodic paralysis and paramyotonia congenita, distinct neuromuscular disorders both of which are thought to involve mutation at SCN4A. Our data confirm the genetic linkage of both disorders with SCN4A. Haplotype analysis also indicates the strong likelihood of allelic heterogeneity in both disorders.     

Studies on the mechanism of action of acetazolamide in the prophylaxis of hyperkalemic periodic paralysis.

Levels of glycogen and cyclic 3′, 5′-adenosine monophosphate (cAMP) were determined in livers of rats treated with 10, 25, 50 or 100 mg/kg of acetazolamide (Diamox). When compared with livers of untreated rats, there were significant decreases in liver glycogen content and significant increases in cAMP levels at all doses of the drug. When liver slices were incubated in the presence of 10^?5 to 10^?3 molar acetazolamide, no difference was found between treated and untreated slices.Plasma insulin and blood glucose levels were also determined and it was found that although plasma insulin levels were significantly increased at all four doses of acetazolamide, blood glucose remained unchanged.These data suggest that acetazolamide induces glycogenolysis through an indirect mechanism dependent upon the release of some endogenous factor, e.g., glucagon or epinephrine, which, in turn, increases levels of cAMP. However, because insulin levels are increased, the increased glycogenolysis does not elevate blood glucose. Thus, it is suggested that acetazolamide stabilizes blood glucose levels while stimulating insulin secretion to potentiate the movement of potassium across muscle membranes and thereby correct the defect which causes attacks of hyperkalemic periodic paralysis.     

Linkage of hyperkalaemic periodic paralysis in Quarter horses to the horse adult skeletal muscle sodium channel gene

A genetic disease observed in certain Quarter horses is hyperkalaemic periodic paralysis (HYPP). This disease causes attacks of paralysis which can be induced by ingestion of potassium. Recent studies have shown that HYPP in humans is due to single base changes within the adult skeletal muscle sodium channel gene. A large Quarter horse pedigree segregating dominant HYPP was studied to determine if mutations of the sodium channel gene are similarly responsible for HYPP in horses. We used cross-species, PCR-mediated, cDNA cloning and sequencing of the horse adult skeletal muscle sodium channel alpha-subunit gene to identify a polymorphism, and then used this polymorphism to see if the horse sodium channel gene was genetically linked to HYPP in horses. The sodium channel gene was indeed found to be tightly linked to HYPP (LOD = 2.7, theta = 0). Our results suggest that HYPP in horses involves the same gene as the clinically similar human disease, and indicates that these are homologous disorders. The future identification of the specific sodium channel mutation causing HYPP in Quarter horses will permit the development of accurate molecular diagnostics of this condition, as has been recently shown for humans.     

Na+,K+-pump stimulation improves contractility in isolated muscles of mice with hyperkalemic periodic paralysis

In patients with hyperkalemic periodic paralysis (HyperKPP), attacks of muscle weakness or paralysis are triggered by K(+) ingestion or rest after exercise. Force can be restored by muscle work or treatment with β(2)-adrenoceptor agonists. A missense substitution corresponding to a mutation in the skeletal muscle voltage-gated Na(+) channel (Na(v)1.4, Met1592Val) causing human HyperKPP was targeted into the mouse SCN4A gene (mutants). In soleus muscles prepared from these mutant mice, twitch, tetanic force, and endurance were markedly reduced compared with soleus from wild type (WT), reflecting impaired excitability. In mutant soleus, contractility was considerably more sensitive than WT soleus to inhibition by elevated [K(+)](o). In resting mutant soleus, tetrodotoxin (TTX)-suppressible (22)Na uptake and [Na(+)](i) were increased by 470 and 58%, respectively, and membrane potential was depolarized (by 16 mV, P < 0.0001) and repolarized by TTX. Na(+),K(+) pump-mediated (86)Rb uptake was 83% larger than in WT. Salbutamol stimulated (86)Rb uptake and reduced [Na(+)](i) both in mutant and WT soleus. Stimulating Na(+),K(+) pumps with salbutamol restored force in mutant soleus and extensor digitorum longus (EDL). Increasing [Na(+)](i) with monensin also restored force in soleus. In soleus, EDL, and tibialis anterior muscles of mutant mice, the content of Na(+),K(+) pumps was 28, 62, and 33% higher than in WT, respectively, possibly reflecting the stimulating effect of elevated [Na(+)](i) on the synthesis of Na(+),K(+) pumps. The results confirm that the functional disorders of skeletal muscles in HyperKPP are secondary to increased Na(+) influx and show that contractility can be restored by acute stimulation of the Na(+),K(+) pumps. Calcitonin gene-related peptide (CGRP) restored force in mutant soleus but caused no detectable increase in (86)Rb uptake. Repeated excitation and capsaicin also restored contractility, possibly because of the release of endogenous CGRP from nerve endings in the isolated muscles. These observations may explain how mild exercise helps locally to prevent severe weakness during an attack of HyperKPP.     

A Na+ channel mutation linked to hypokalemic periodic paralysis exposes a proton-selective gating pore

The heritable muscle disorder hypokalemic periodic paralysis (HypoPP) is characterized by attacks of flaccid weakness, brought on by sustained sarcolemmal depolarization. HypoPP is genetically linked to missense mutations at charged residues in the S4 voltage-sensing segments of either CaV1.1 (the skeletal muscle L-type Ca(2+) channel) or NaV1.4 (the skeletal muscle voltage-gated Na(+) channel). Although these mutations alter the gating of both channels, these functional defects have proven insufficient to explain the sarcolemmal depolarization in affected muscle. Recent insight into the topology of the S4 voltage-sensing domain has aroused interest in an alternative pathomechanism, wherein HypoPP mutations might generate an aberrant ionic leak conductance by unblocking the putative aqueous crevice ("gating-pore") in which the S4 segment resides. We tested the rat isoform of NaV1.4 harboring the HypoPP mutation R663H (human R669H ortholog) at the outermost arginine of S4 in domain II for a gating-pore conductance. We found that the mutation R663H permits transmembrane permeation of protons, but not larger cations, similar to the conductance displayed by histidine substitution at Shaker K(+) channel S4 sites. These results are consistent with the notion that the outermost charged residue in the DIIS4 segment is simultaneously accessible to the cytoplasmic and extracellular spaces when the voltage sensor is positioned inwardly. The predicted magnitude of this proton leak in mature skeletal muscle is small relative to the resting K(+) and Cl(-) conductances, and is thus not likely to fully account for the aberrant sarcolemmal depolarization underlying the paralytic attacks. Rather, it is possible that a sustained proton leak may contribute to instability of V(REST) indirectly, for instance, by interfering with intracellular pH homeostasis.     

Genetic homogeneity of Pelizaeus-Merzbacher disease: tight linkage to the proteolipoprotein locus in 16 affected families. PMD Clinical Group.

Among the numerous leukodystrophies that have an early onset and no biochemical markers, Pelizaeus-Merzbacher disease (PMD) is one that can be identified using strict clinical criteria and demonstrating an abnormal formation of myelin that is restricted to the CNS in electrophysiological studies and brain magnetic resonance imaging (MRI). In PMD, 12 different base substitutions and one total deletion of the genomic region containing the PLP gene have been reported, but, despite extensive analysis, PLP exon mutations have been found in only 10%-25% of the families analyzed. To test the genetic homogeneity of this disease, we have carried out linkage analysis with polymorphic markers of the PLP genomic region in 16 families selected on strict diagnostic criteria of PMD. We observed a tight linkage of the PMD locus with markers of the PLP gene (cDNA PLP, exon IV polymorphism) and of the Xq22 region (DXS17, DXS94, and DXS287), whereas the markers located more proximally (DXYS1X and DXS3) or distally (DXS11) were not linked to the PMD locus. Multipoint analysis gave a maximal location score for the PMD locus (13.98) and the PLP gene (8.32) in the same interval between DXS94 and DXS287, suggesting that in all families PMD is linked to the PLP locus. Mutations of the extraexonic PLP gene sequences or of another unknown close gene could be involved in PMD. In an attempt to identify molecular defects of this genomic region that are responsible for PMD, these results meant that RFLP analysis could be used to improve genetic counseling for the numerous affected families in which a PLP exon mutation could not be demonstrated.     

Exclusion of linkage between hypokalemic periodic paralysis (HOKPP) and three candidate loci.

Hypokalemic periodic paralysis (HOKPP) is an autosomal dominant neuromuscular disorder characterized by flaccid paralysis accompanied by lowered serum potassium levels. We have tested polymorphic markers linked to the adult skeletal muscle sodium channel (SCN4A) locus at 17q23-q25, the T-cell receptor beta (TCRB) locus at 7q35, and the H-Ras cellular proton-cogene locus (HRAS) at 11p15.5 for linkage with the affected phenotype in a single multigenerational pedigree. No evidence for genetic linkage to HOKPP was found at any of the candidate loci.     

Evidence for linkage on chromosome 3q25-27 in a large autism extended pedigree

Though autism shows strong evidence for genetic etiology, specific genes have not yet been found. We tested for linkage in a candidate region on chromosome 3q25-27 first identified in Finnish autism families [1]. The peak in this previous study was at D3S3037 (183.9 cM). We tested this region in seven affected family members and 24 of their relatives from a single large extended Utah pedigree of Northern European ancestry. A total of 70 single nucleotide polymorphisms (SNPs) were analyzed from 165 to 204 cM. The maximum NPL-all nonparametric score using SimWalk2snp was 3.53 (empirical p val ue = 0.0003) at 185.2 cM (SNP rs1402229), close to the Finnish peak. A secondary analysis using MCLINK supported this result, with a maximum of 3.92 at 184.6 cM (SNP rs1362645). We tested for alterations in a candidate gene in this region, the fragile X autosomal homolog, FXR1. No variants likely to contribute to autism were found in the coding sequence, exon-intron boundaries, or the promoter region of this gene.     

Gating defects of a novel Na+ channel mutant causing hypokalemic periodic paralysis

Hypokalemic periodic paralysis type 2 (hypoPP2) is an inherited skeletal muscle disorder caused by missense mutations in the SCN4A gene encoding the alpha subunit of the skeletal muscle Na+ channel (Nav1.4). All hypoPP2 mutations reported so far target an arginine residue of the voltage sensor S4 of domain II (R672/G/H/S). We identified a novel hypoPP2 mutation that neutralizes an arginine residue in DIII-S4 (R1132Q), and studied its functional consequences in HEK cells transfected with the human SCN4A cDNA. Whole-cell current recordings revealed an enhancement of both fast and slow inactivation, as well as a depolarizing shift of the activation curve. The unitary Na+ conductance remained normal in R1132Q and in R672S mutants, and cannot therefore account for the reduction of Na+ current presumed in hypoPP2. Altogether, our results provide a clear evidence for the role of R1132 in channel activation and inactivation, and confirm loss of function effects of hypoPP2 mutations leading to muscle hypoexcitability.     

In vivo and in vitro sodium pump activity in subjects with thyrotoxic periodic paralysis.

OBJECTIVE--To examine whether sodium pump activity plays a part in the pathogenesis of thyrotoxic periodic paralysis. DESIGN--Measurement of platelet sodium-potassium ATPase and in vivo sodium pump activities in healthy subjects and thyrotoxic subjects with and without paralysis. SETTING--University hospital in Hong Kong. SUBJECTS--21 healthy subjects, 23 untreated thyrotoxic subjects, 13 untreated men with periodic paralysis, seven treated thyrotoxic subjects, and six treated men with periodic paralysis. MAIN OUTCOME MEASURES--Platelet Na+, K(+)-ATPase activity and plasma rubidium concentration after oral loading. RESULTS--Median (range) platelet Na+, K(+)-ATPase activity in thyrotoxic subjects was 253 (169-821) mumol inorganic phosphate/h/g protein--significantly higher than that in healthy subjects (134 (81-180) mumol/h/g protein; p less than 0.001). Na+, K(+)-ATPase activity in those with periodic paralysis was 374 (195-1196) mumol/h/g protein, again significantly higher than that in healthy subjects (p less than 0.001) and that in other thyrotoxic subjects (p less than 0.01) despite similar degrees of hyperthyroidism. Activities in treated thyrotoxic subjects with and without periodic paralysis were 148 (110-234) and 131 (86-173) mumol/h/g protein respectively. Mean (95% confidence interval) plasma rubidium concentration five hours after oral administration in thyrotoxic subjects (7.0 (6.6 to 7.5) mumol/l) was significantly lower than in healthy subjects (10.2 (9.5 to 10.9) mumol/l; p less than 0.001) and higher than in those with periodic paralysis (6.0 (5.7 to 6.3) mumol/l; p less than 0.01). CONCLUSIONS--Sodium pump activity in untreated subjects with periodic paralysis is higher than in other thyrotoxic subjects, and this may be responsible for the hypokalaemia.     

The role of pedigree information in combined linkage disequilibrium and linkage mapping of quantitative trait loci in a general complex pedigree

Combined linkage disequilibrium and linkage (LDL) mapping can exploit historical as well as recent and observed recombinations in a recorded pedigree. We investigated the role of pedigree information in LDL mapping and the performance of LDL mapping in general complex pedigrees. We compared using complete and incomplete genotypic data, spanning 5 or 10 generations of known pedigree, and we used bi- or multiallelic markers that were positioned at 1- or 5-cM intervals. Analyses carried out with or without pedigree information were compared. Results were compared with linkage mapping in some of the data sets. Linkage mapping or LDL mapping with sparse marker spacing ( approximately 5 cM) gave a poorer mapping resolution without considering pedigree information compared to that with considering pedigree information. The difference was bigger in a pedigree of more generations. However, LDL mapping with closely linked markers ( approximately 1 cM) gave a much higher mapping resolution regardless of using pedigree information. This study shows that when marker spacing is dense and there is considerable linkage disequilibrium generated from historical recombinations between flanking markers and QTL, the loss of power due to ignoring pedigree information is negligible and mapping resolution is very high.     

A comprehensive linkage analysis of chromosome 21q22 supports prior evidence for a putative bipolar affective disorder locus.

Previously, we demonstrated evidence of linkage to bipolar affective disorder (BP) in a single large, multigenerational family with a LOD score of 3.41 at the PFKL locus on chromosome 21q22.3. Additional families showed little support for linkage to PFKL under homogeneity or heterogeneity, in that study. We have expanded on that analysis, with 31 microsatellite markers at an average marker spacing of </=2 cM, in the largest multigenerational BP pedigree series reported to date. A two-point heterogeneity (alpha=0.5) LOD score of 3.35 (P<.000156) was found at the D21S1260 locus, 5 cM proximal to PFKL. Polylocus analysis with a cluster of three neighboring markers was consistent with these results (PL-HetLOD = 3.25). In the design of this study, 373 individuals from 40 families (from a total set of 1,508 individuals in 57 families) were chosen, as a cost-effective approach to genotyping this large sample set. Linkage analyses were performed with an "affecteds-only" method. As such, our results are based solely on genetic information from affected individuals, without assumptions about the disease-locus genotypes of the unaffecteds. Furthermore, for ease of comparison, this study was performed with the same approach as a 10-cM genome scan for BP loci, the results of which will be reported elsewhere.     

Estimating linkage disequilibrium between a polymorphic marker locus and a trait locus in natural populations.

Positional cloning of gene(s) underlying a complex trait requires a high-resolution linkage map between the trait locus and genetic marker loci. Recent research has shown that this may be achieved through appropriately modeling and screening linkage disequilibrium between the candidate marker locus and the major trait locus. A quantitative genetics model was developed in the present study to estimate the coefficient of linkage disequilibrium between a polymorphic genetic marker locus and a locus underlying a quantitative trait as well as the relevant genetic parameters using the sample from randomly mating populations. Asymptotic covariances of the maximum-likelihood estimates of the parameters were formulated. Convergence of the EM-based statistical algorithm for calculating the maximum-likelihood estimates was confirmed and its utility to analyze practical data was exploited by use of extensive Monte-Carlo simulations. Appropriateness of calculating the asymptotic covariance matrix in the present model was investigated for three different approaches. Numerical analyses based on simulation data indicated that accurate estimation of the genetic parameters may be achieved if a sample size of 500 is used and if segregation at the trait locus explains not less than a quarter of phenotypic variation of the trait, but the study reveals difficulties in predicting the asymptotic variances of these maximum-likelihood estimates. A comparison was made between the statistical powers of the maximum-likelihood analysis and the previously proposed regression analysis for detecting the disequilibrium.     

Paroxysmal dystonic choreoathetosis: tight linkage to chromosome 2q.

Paroxysmal dystonic choreoathetosis (PDC) is characterized by attacks of involuntary movements that last up to several hours and occur at rest both spontaneously and following caffeine or alcohol consumption. We analyzed a Polish-American kindred with autosomal dominant PDC and identified tight linkage between the disorder and microsatellite markers on chromosome 2q (maximum two-point LOD score 4.77; recombination fraction 0). Our results clearly establish the existence of a locus for autosomal dominant PDC on distal chromosome 2q. The fact that three other paroxysmal neurological disorders (periodic ataxia with myokymia and hypo- and hyperkalemic periodic paralysis) are due to mutation in ion-channel genes raises the possibility that PDC is also due to an ion-channel gene mutation. It is noteworthy that a cluster of sodium-channel genes is located on distal chromosome 2q, near the PDC locus. Identifying the PDC locus on chromosome 2q will facilitate discovery of the PDC gene and enable investigators to determine whether PDC is genetically homogeneous and whether other paroxysmal movement disorders are also genetically linked to the PDC locus.