January 1, 2019;
Myasthenic congenital myopathy from recessive mutations at a single residue in NaV1.4.
OBJECTIVE: To identify the genetic and physiologic basis for recessive myasthenic congenital myopathy in 2 families, suggestive of a channelopathy involving the sodium channel gene, SCN4A
METHODS: A combination of whole exome sequencing and targeted mutation analysis, followed by voltage-clamp studies of mutant sodium channels expressed in fibroblasts (HEK cells) and Xenopus oocytes.
RESULTS: Missense mutations of the same residue in the skeletal muscle
sodium channel, R1460 of NaV1
.4, were identified in a family and a single patient of Finnish origin (p.R1460Q) and a proband in the United States (p.R1460W). Congenital hypotonia, breathing difficulties, bulbar weakness, and fatigability had recessive inheritance (homozygous p.R1460W or compound heterozygous p.R1460Q and p.R1059X), whereas carriers were either asymptomatic (p.R1460W) or had myotonia (p.R1460Q). Sodium currents conducted by mutant channels showed unusual mixed defects with both loss-of-function (reduced amplitude, hyperpolarized shift of inactivation) and gain-of-function (slower entry and faster recovery from inactivation) changes.
CONCLUSIONS: Novel mutations in families with myasthenic congenital myopathy have been identified at p.R1460 of the sodium channel. Recessive inheritance, with experimentally established loss-of-function, is a consistent feature of sodium channel based myasthenia, whereas the mixed gain of function for p.R1460 may also cause susceptibility to myotonia.
sodium channel activity
Disease Ontology terms:
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Figure 1. Sodium channel mutations(A) Segregation of clinical phenotype and genotype among 7 carriers of p.R1460Q in family 1 from Finland. (B) Location of p.R1460 in the pore-forming subunit (NaV1.4) along with established sites for sodium channelopathies of skeletal muscle. CMS = congenital myasthenic syndrome; HyperPP = hyperkalemic periodic paralysis; HypoPP = hypokalemic periodic paralysis; PAM = paramyotonia congenita; SCM = sodium channel myotonia.
Figure 2. Sodium current density was reduced for cells transfected with R1460 mutant constructs, but the voltage dependence of activation was not altered(A) Traces show sodium currents elicited by depolarization from −120 mV to test potentials between −75 and +60 mV. Note the higher gain for the scale bars in the mutant channel traces. (B) Peak current density is shown as a function of test potential and displays a marked reduction in current amplitude for both mutants. (C) The voltage dependence of channel activation, as shown by relative conductance, was indistinguishable between the wild-type (WT) and mutant channels. Black squares, blue triangles, and red circles correspond to WT (n = 16), R1460Q (n = 12), and R1460W (n = 16), respectively.
Figure 3. Fast inactivation of R1460 mutant channels had both gain-of-function and loss-of-function defects(A) The steady-state voltage dependence of fast inactivation was shifted leftward (hyperpolarized) for both R1460 mutant channels relative to wild-type (WT). Inset shows the voltage protocol used to measure inactivation produced by a 300 ms conditioning pulse at various potentials. (B) The rate of inactivation was slower for R1460 mutant channels at depolarized potentials, as shown by the superposition of amplitude normalized currents elicited at 10 mV. (C) Recovery from fast inactivation was faster for both R1460Q and R1460W, compared to WT channels (tau recovery of 4.7 ± 0.6 ms, p <0.0001 [n = 5]; 6.4 ± 0.6 ms, p <0.0001 [n = 7]; 16.8 ± 0.4 ms [n = 4], respectively). Data show the time course for the recovery of peak current amplitude at a holding potential of −80 mV, after channels were inactivated with a conditioning pulse of 30 ms at −10 mV (inset). (D) Plot summarizing the voltage-dependent kinetics for entry to or recovery from fast inactivation. Three separate protocols were used to measure inactivation kinetics, over the entire voltage range (see text, Results). Overall, the changes in steady-state fast inactivation (A) are loss of function, whereas the slower entry and more rapid recovery from fast inactivation are gain-of-function changes.
Figure 4. The use-dependent reduction in sodium current peak amplitude was less pronounced in R1460 mutants than in wild-type (WT) channelsData show relative peak sodium current elicited by 2 ms depolarizing pulses to 10 mV applied at 60 Hz (A) or 100 Hz (B), from a holding potential of −80 mV.
Figure 5. Slow inactivation was not altered by either R1460W or R1460Q(A) Onset of slow inactivation showed identical kinetics between wild-type (WT) and mutant channels. Inset shows the voltage protocol to characterize the onset of slow inactivation by stepping to −10 mV for a variable duration (entry time), and then measuring the decline in relative loss current that fails to recover within 20 ms at −120 mV. (B) The rate of recovery from slow inactivation was comparable between WT and R1460 mutant channels. Channels were slow inactivated by a 30-second step to −10 mV (inset), and the data show recovery as the relative increase in current after repolarizing to −80 mV for a variable duration (recovery time). (C) The voltage dependence of steady-state slow inactivation was indistinguishable between WT and R1460 mutant channels. Thirty-second conditioning pulses to various conditioning potentials (inset) were used to determine the voltage dependence of inactivation. Smooth curves show best fits to the data with V1/2 of −59.1 ± 1.9 mV for WT, −60.9 ± 2.8 mV for R1460Q, and −63.4 ± 2.6 mV for R1460W. The number of cells for the mean values shown in the data was 5–8 for WT and both R1460 mutant channels.
Figure 6. The R1460 mutations did not cause a gating pore leakage currentThe steady-state current (without subtraction of the nonspecific leak) recorded from oocytes expressing high levels of wild-type (WT) or R1460 mutant channels is plotted as a function of the membrane potential. Tetrodotoxin was added to block sodium currents through the main pore. The I–V relation was identical for WT and R1460 mutant channels, while the positive control for the p.R1132Q mutation in hypokalemic periodic paralysis showed a gating pore leakage current (large negative current for membrane potentials more negative than −80 mV). Data are from 6 to 8 oocytes per construct.
Defective fast inactivation recovery of Nav 1.4 in congenital myasthenic syndrome.