Identification of Mutation in SCN4A, Hot-Spot for Periodic Paralyses, in a Large Chinese Family with a Typical Normokalemic PP Using the Whole-Exome Sequencing

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Introduction
Periodic paralysis (PP) is a group of ion-channel dysfunction diseases [1]. Based on serum potassium level, PP can be classified into hypokalemic periodic paralysis (Hypo KPP), hyperkalemic periodic paralysis (Hyper KPP), and normokalemic periodic paralysis (Normo KPP). The term Normo KPP was originally used in the 1960s [2] to describe a very rare disease characterized by repeated attacks of flaccid muscle weakness or paralysis, despite normal serum potassium levels. Trigger factors for these episodes include rest after acute exercise, cold, hunger, emotional tension, carbohydrate-rich meals, and potassium supplements. Concerns episodes of paralysis is most in the early morning or during breaks from vigorous activities [3,4]. Several familial and sporadic cases of this disease have been reported [5,6]. Pathogenic mutations in exons 12, 13, 18, and 24 of SCN4A, leading to the amino acid changes T704M, R675G, R675W, R675H, R675Q, R1129Q, and M1592V, have been reported to cause Normo KPP in patients from China and other countries [5][6][7][8] . Compared with other diseases causing PP, Normo KPP has no specific clinical features, with electromyography, electrocardiogram, blood sugar, cerebrospinal fluid, and routine biochemical laboratory and auxiliary examinations generating normal results during an attack.
In this study, we adopted the Ion Torrent Proton sequencing platform for whole-exome scanning, which has advantages over Sanger sequencing in terms of read throughput, cost, operation time, and potential for use as a standardized procedure in hospital.
Given the diverse clinical symptoms of Normo KPP, the scope of the investigation was expanded to the whole-exome scale, to obtain comprehensive information on the genetics underlying the pathogenic features of the condition. We characterized a Chinese family with atypical Normo KPP features and identified a classical c.2111C>T (p.T704M) mutation in SCN4A that is predicted to be responsible for the disease. performance of electromyogram showed no myotonic discharge or increased insertion activity. The patients awake paralyzed the morning following vigorous exercise and cold temperatures.

Patients and Families
Significantly atypical feature from other Normo KPP families [6][7][8][9] was that treatment with large doses of physiological saline had no effect, whereas calcium gluconate was effective. In addition, acetazolamide could reduce the number of attacks and alleviate attack symptoms.

Bioinformatics Analyses
Raw data from Ion Torrent Proton runs were processed using the Ion Torrent platform-specific pipeline software, Torrent Suite v4.6 (Life Technologies) to generate sequence reads, for sequence alignment, and to extract single nucleotide variants and indels. Initial variant calling was performed using the Ion Torrent platform-specific pipeline software with the plug-in ''variant caller" program. Two annotation steps were used to obtain information about gene mutations associated with disease. Torrent Suite Variant Caller (TSVC; version 4.6) plug-in generated files were filtered and annotated using ANNOVAR software [10], then the variant caller format (VCF) files generated by ANNOVAR were further filtered and annotated using WANNOVAR software [11].
LOVD database and PubMed literature queries were performed to determine whether variants had been reported as pathogenic and identify disease loci; suspected pathogenic mutations were verified by Sanger sequencing. SCN4A sequences of five non-human species for homology comparisons were obtained using NCBI BLAST and alignments were carried out using Clustal W to check conservation. and Xenopus ( Figure 2D).

Discussion
SCN4A encodes the a-subunit, voltage-gated sodium channel, Nav1.4 whose structure consists of four domains (DI-DIV) of six transmembrane helical segments (S1-S6). Here, we report a pathological mutation in the SCN4A gene in a family presenting with hereditary Normo KPP. In the present study, whole-exome and Sanger sequencing identified C/T heterozygosity in the SCN4A gene at nucleotide position 2111 in the 13th exon. Both of the affected members in the family carried the p.T704M mutation, whereas the unaffected members did not. Pathophysiological studies show the mutation conduct gating pore current in both activated and slowinactivated states, which would cause increased influx of sodium near the resting membrane potential, membrane depolarization, sodium overload, action potential failure [12]. Structural studies elucidate the molecular mechanism by which mutations in S4 cause pathogenic gating pore currents and suggest that ion permeation through the gating pore is controlled dynamically by the state of the voltage sensor and by rotamer conformations of R4 [13]. which mutations are a rare cause of hypokalaemic or hyperkalaemic periodic paralysis, is an example of a possible modifying gene [19].
It now appears that Ca 2+ activated-K + -(BK) channel encoded by one gene (slo1/KCNMA1) may have relevance in disorders associated with abnormal K + ion homeostasis including periodic paralysis and myotonia [20,21].
Saline solution is first choice for acute management of Normo KPP. The dose of saline solution usually needs more than three thousand milliliter a day and patiens with NormoKPP regularly recovered within 2-3 days. Patients of the family with Normo KPP showed no response to saline solution, intravenous calcium gluconate was chosen to treat patients according to protocol of Neurology textbook [22].We presumed the mechanism of which was calcium gluconate enhance opening of calcium-activated K channels [20,21]. The prophylactic therapy with diuretics is effective in treating Normo KPP. Acetazolamide was empiric treatment for NormoKPP, the mechanism of action is unclear.
Another diuretics hydrochlorothiazide was confirmed being effective in the prophylactic treatment of normoKPP caused by the SCN4A mutation of p. Thr704Met [23]. The results of this study demonstrate potential new clinical features of PP associated with p.T704M mutation in SCN4A. In addition, we also demonstrate the feasibility of next generation sequencing for mutation detection in a complex monogenic disease.