Luo et al. BMC (2019) 19:92 https://doi.org/10.1186/s12883-019-1322-6

RESEARCH ARTICLE Open Access Identification of gene mutations in patients with primary periodic using targeted next-generation sequencing Sushan Luo1†, Minjie Xu2†, Jian Sun1, Kai Qiao3, Jie Song1, Shuang Cai1, Wenhua Zhu1, Lei Zhou1, Jianying Xi1, Jiahong Lu1, Xiaohua Ni2, Tonghai Dou4 and Chongbo Zhao1,5*

Abstract Background: Primary is characterized by recurrent quadriplegia typically associated with abnormal serum potassium levels. The molecular diagnosis of primary PP previously based on Sanger sequencing of hot spots or exon-by-exon screening of the reported genes. Methods: We developed a gene panel that includes 10 -related genes and 245 - and -related genes and used this panel to diagnose 60 patients with primary periodic paralysis and identify the disease-causing or risk-associated gene mutations. Results: Mutations of 5 genes were discovered in 39 patients (65.0%). SCN4A, KCNJ2 and CACNA1S variants accounted for 92.5% of the patients with a genetic diagnosis. Conclusions: Targeted next-generation sequencing offers a cost-effective approach to expand the genotypes of primary periodic paralysis. A clearer genetic profile enables the prevention of paralysis attacks, avoidance of triggers and the monitoring of complications. Keywords: Primary periodic paralysis, Targeted next-generation sequencing, Gene panel, Gene mutation distribution, Calcium homeostasis.

Background (SCN4A) and potassium voltage-gated channel subfamily Periodic paralysis (PP) is characterized by episodes of J member 2 (KCNJ2), that encode voltage-gated chan- muscle weakness that occur at irregular intervals due to nels in muscle membranes that generate or sustain ion . This highly hetero- membrane potentials [1]. Furthermore, missense muta- geneous group of muscle diseases can be further divided tions in Ryanodine receptor type 1 (RYR1), which dir- into primary and secondary disorders. The characteris- ectly couples with a voltage-gated L-type Ca2+ channel tics of primary PP are that they are genetic disorders, (dihydropyridine receptor, DHPR) to generate usually presenting before the age of 20, often with more excitation-contraction and promote the rapid and gener- than one affected generation and with the exclusion of alized release of calcium within myofibrils, were recently other diseases that can alter serum potassium levels. The identified in patients with PP [2, 3]. most common genetic causes of primary PP include Prior to the development of next-generation sequen- genes, such as calcium voltage-gated channel subunit cing (NGS), the molecular diagnosis of primary PP had alpha 1S (CACNA1S), sodium channel α subunit been based on Sanger sequencing of hot spots or exon-by-exon screening of the reported genes. This ap- * Correspondence: [email protected] proach is time-consuming and, most importantly, is con- †Sushan Luo and MinjieXu contributed equally to the study. fined to the known genes and thus is less likely to 1 Department of Neurology, Huashan Hospital, Fudan University, Shanghai identify cases with more complicated genetic etiologies. 200040, China 5Department of Neurology, Jing’an District Center Hospital of Shanghai, The targeted NGS technique has been employed to es- Shanghai 200040, China tablish molecular diagnosis in patients with primary Full list of author information is available at the end of the article

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Luo et al. BMC Neurology (2019) 19:92 Page 2 of 8

and muscular dystrophies during the past 5 following criteria: 1) age of onset ≤35 years; 2) recurrent years [4–6]. Targeted NGS is considered as a muscle weakness ≥2 times; and 3) a significant decline cost-effective strategy for muscle disorders with hetero- in the compound muscle action potential (CMAP) amp- geneous genetic causes and allows us to broaden the litude ≥30% of the baseline value in long exercise test phenotypic spectrum of hereditary myopathies based on (LET) (Fig. 1). The standard procedure of LET was pre- newly identified mutations. viously described [7]. Serum potassium levels during the Here, we described the application of a targeted attacks either decrease, remain at a normal level (3.5– NGS panel that includes 10 ion channel-related genes 5.5 mmol/L) or increase. Patients with , and 245 muscular dystrophy and myopathy-related adrenoid gland dysfunction and renal tubular acidosis genes to obtain a genetic diagnosis in a cohort of 60 are excluded in this study. Taken together, we included a Chinese Han patients with clinically diagnosed pri- total of 60 patients from 53 pedigrees for further genetic mary PP. screening with NGS.

Methods Targeted next-generation sequencing Patient ascertainment Genomic DNA was extracted from peripheral blood This study was approved under the guidelines of the In- using the High Pure PCR. stitutional Ethics Committee of the Huashan Hospital, Template Preparation Kit (Roche, Basel, CH) according and conducted according to the principles in the Declar- to the manufacturer’s instructions. The DNA fragments ation of Helsinki. Written Informed consent was ob- were enriched by performing solution-based hybridization tained from each participant for providing the clinical capture, followed by sequencing using an IlluminaMiseq information and bio-sample for further analysis. For platform (Illumina, San Diego, CA, USA) with the 2 × 300 those participants under the age 18, the written consent bp paired-end read module. The hybridization capture was signed by their parents. The recruitment of patients procedure was performed using the SureSelect Library with clinically defined primary PP was based on the Prep Kit (Agilent, Santa Clara, CA, USA). The defined

Fig. 1 Inclusion and diagnostic strategy for targeted NGS in patients with primary PP. TPP: thyrotoxic periodic paralysis. CMAP: compound muscle action potential Luo et al. BMC Neurology (2019) 19:92 Page 3 of 8

target regions included 5060 regions containing 255 genes products were sequenced on an ABI3730xl DNA known to cause muscle disorders, including ion Analyzer (Applied Biosystems). A genotype-phenotype channelopathies, limb-girdle muscular dystrophies, co-segregation analysis was performed if the parents’ Duchenne/Becker muscular dystrophies, congenital blood samples were available. To detect splicing muta- muscular dystrophies, metabolic myopathies, congenital my- tions, RNA was extracted from frozen muscle biopsies opathies and distal myopathies (Additional file 1 Table S1). using RNAiso Plus (Takara), and cDNA was synthetized The oligonucleotides covered all coding exons, UTR regions using a PrimeScriptTMRT Reagent Kit (Takara). and intron-exon boundaries, including at least 10 intronic nucleotides. The DNA was first quantified using Qubit 2.0 Results (Thermo Fisher Scientific, Waltham, MA, USA). In total, Sequence reads and average coverage of the targeted 3 μg of genomic DNA were sheared by sonication using a regions DiangenodeBioruptor® Plus and hybridized using Biotinyl- We performed targeted NGS in 60 patients with clinic- ated RNA oligonucleotide baits. The captured fragments ally defined primary periodic paralysis from 53 families were removed from the solution using streptavidin-coated to identify 17 known variants and 8 novel mutations in magnetic beads (Dynabeads® MyOne™ Streptavidin T1, skeletal muscle ion channel genes (Additional file 2: Thermo Fisher Scientific) and subsequently eluted. The Table.S1). These variants included 2 pathogenic, 16 resulting libraries were quantified by qPCR before proceed- likely pathogenic and 7 variants with uncertain signifi- ing to the Illumina Miseq platform. cance. No CNVs have been found in these cases. On average, 3625 raw variants were detected per pa- Variant analysis and interpretation tients, and 3154 high-quality variants remained after fil- After the Miseq sequencing, the raw reads from each tering. The average sequencing depth was 488.11×, and sample were sorted according to the index sequences. the average coverage was 99.45%. GC-rich sequences The adapter sequences were trimmed using cutadapt have been shown to reduce the efficiency of the probe (http://code.google.com/p/cutadapt/). SolexaQA was capture. Ion channel genes with GC-rich (> 70%) target used to remove the low-quality bases (< Q20). The clean regions are listed in Additional file 2: Table. S2. Within reads were aligned to the human reference genome these regions, a much lower sequencing depth and (hg19) using the Burrows–Wheeler Aligner (BWA; ver. coverage have been observed. 0.7.11) [8–10]. After the alignment, the PCR duplicates were removed using the Picard tools (ver. 1.109) Mark Variants with established skeletal muscle ion channel- Duplicates package. The realignment around the known related genes indel sites and Base Quality Score Recalibration (BQSR) Variants of established skeletal muscle ion channel were performed using GATK (ver. 3.3) [11]. GATK Hap- genes, including CACNA1S, SCN4A and KCNJ2, were lotypeCaller was used to call the raw variants. The indels identified in a total of 37 patients (37/60, 61.67%)(Fig. 2). and SNPs were annotated using ANNOVAR [12]. Public SCN4A (NM_000334) mutations accounted for the ma- databases, including dbSNP138, 1000 Genome project, jority in this primary PP cohort (19/39,48.7%). We iden- Exome Sequencing Project, ClinVar and HGMD [13], tified 10 SCN4A variants including 6 reported mutations were used to screen the variants. The functional effect (c.2014C > T p.Arg672Cys, c.2024G > A p.Arg675Gln, prediction was evaluated using the PolyPhen-2 and SIFT c.2111C > T p.Thr704Met, c.4352G > T p.Arg1451Leu, scores [14, 15]. To detect the copy number variant c.4774A > G p.Met1592Val, and c.5293G > A (CNVs), the sequencing depth of each region covered by p.Ala1765Thr) and 4 novel mutations (c.107_109del the probes was calculated according to the alignment p.Glu36del, c.121C > T p.Arg41Trp, c.718G > A files. The ExomeDepth [15] package was also used to p.Val240Met, and c.3868 T > C p.Phe1290Leu) (Fig. 3a). identify potential CNVs. Confirmed point mutation sam- Among them, c.2024G > A p.Arg675Gln was identified ples in the same sequence run served as controls in the in 8 patients from 6 families as a potential hotspot. Two CNV analysis. All variants are classified in American variants with uncertain significance (c.107_109del College of Medical Genetics and Genomics standards p.Glu36del and c.5293G > A p.Ala1765Thr) are distrib- and guidelines [16]. uted in the N′- terminus and C′-terminusof the α-subunit, which are distant from the 24 α-helical trans- Variants verification membrane segments. To further verify the candidate mutations, we performed Second most prevalent gene responsible for primary Sanger sequencing of the DNA samples extracted from periodic paralysis was KCNJ2 (NM_000891). We the patients and their family members. PCR was per- identified 8 reported variants (c.199C > T p.Arg67Trp, formed using GoldStarTaq DNA Polymerase (CWbio- c.211G > A p.Asp71Asn, c.566G > T p.Arg189Ser, tech) according to a standard protocol [17]. The PCR c.644G > A p.Gly215Asp, c.652C > T p.Arg218Trp, c. Luo et al. BMC Neurology (2019) 19:92 Page 4 of 8

Fig. 2 Proportions of different gene variants

899G > A p.Gly300Asp, c.919A > G p.Met307Val, and Variants in rarely reported skeletal muscle ion channel- c.921G > C p.Met307Ile) in a total of 14 patients (Fig. 3b). related genes Moreover, we identified 4 likely pathogenic variants One known variant (c.8290G > A p.Glu2764Lys) and one (c.614 T > A p.Phe205Tyr, c.1583G > A p.Arg528His, novel variant (c.12428C > T p.Ala4143Val) were identified c.2965G > A p.Glu989Lys and c.3716G > A p.Arg1239His) in RYR1 gene. The patient harboring heterozygous RYR1 in CACNA1S gene (NM_000069). variant p.Glu2764Lys had repeated episodes of generalized normokalemic paralysis after 20 years old. Another patient carrying heterozygous RYR1 variant p.Ala4143Val com- Clinical features of patients with known skeletal muscle plained of recurrent muscle weakness in proximal lower ion channel-related gene mutations limbs with since 24 years old with a fre- Patients with SCN4A mutations are all male with quency of 2–3 times per year. Neither of these cases had juvenile-onset hypoPP or normoPP. Noticeably, one case proceeding family history of neuromuscular disorders. had normal serum potassium level during attacks (3.8 There is no cognitive, bulbar or respiratory involvement. mmol/L, normal, 3.5–5.5 mmol/L), but the muscle weakness got worsened by steroids administration. We identified a re- Discussion ported homozygous SCN4A mutation (c.4352G > T In this study, we designed a targeted NGS gene assay p.Arg1451Leu) in a teenage boy with hypokalemia [18, 19]. comprising 10 ion channel-related genes and 245 mus- The parents are both heterozygous carriers of the same vari- cular dystrophy/myopathy-related genes to screen pa- ant but only manifest subclinical after EMG study. tients with primary PP who were referred to our A total of 14 patients (14/39, 35.9%) with ATS mani- diagnostic center. In total, we elucidated genetic contri- fested early onset periodic paralysis, variable dysmorphic butions in 65.0% (39/60) of the primary periodic paraly- features and cardiac arrhythmia, except one case whose sis cohort. ECG result is not available. Ventricular arrhythmia includ- Known responsible gene variants (CACNA1S, KCNJ2 ing premature ventricular contraction, bigeminy runs, and SCN4A) were identified in 61.67% of the whole co- couplets, triplets and bidirectional ventricular tachycardia, hort. So far, this is the first cohort to study the frequen- was demonstrated in 13 cases (except P16 who did not cies of known variants in Chinese patients with primary perform ECG study, Additional file 1: Table.S1). Long QT PP, as follows: (1) SCN4A variants are the most common syndrome was documented in 11 cases. genetic cause; (2) KCNJ2 variants are the second most All 4 cases (4/39,10.2%) with CACNA1S mutations common; (3) CACNA1S mutation is relatively rare. are either autosomal dominant inherited (2 patients) Among the 37 cases with HypoPP, SCN4A mutation or sporadic periodic paralysis (2 patients) with hypo- group accounts for 29.73% (11/37), KCNJ2 and CAC- kalemia. Potassium supplement and methazolamide NA1S account for 10.81% (4/37) respectively. While oral administration effectively reduced the episodic CACNA1S mutations are the most common in HypoPP frequency. patients in USA and European population [20–22], Luo et al. BMC Neurology (2019) 19:92 Page 5 of 8

Fig. 3 Distribution of SCN4A and KCNJ2 variants in the Nav1.4 (a) and Kir2.1 structure (b). Red ovals depict novel variants and green ovals are reported variants

SCN4A accounts for the majority of HypoPP across will cause the positively charged S4 segment to move to- Chinese individuals. Since most patients recruited in this wards cell surface and this motion is transferred to the study are adults, a selection bias may exist. Besides, pore domain S5–6 via the linker, abruption of which re- there are different genetic variations for patients with sults in a failed conformational change thus lead to de- primary PP across populations. With regard to the hot layed opening of the sodium channel. Another novel spot identified in this study, a SCN4A variant variant p.Phe1290Leu was located in the N-terminal of (p.Arg675Gln) accounted for the majority of all patients Domain III-S6, the pore-forming segment of the channel. with sodium channelopathies (42.1%, 8/19). The most The remaining 3 novel variants, i.e.,p.36_37del, prevalent sodium channel variants in USA population p.Arg41Trp and p.Ala1765Thr, are distributed in the N′- [21], T704 M and M1592 V, were identified in three terminus and C′-terminus of the α-subunit. Though they Chinese individuals with normokalemic PP. are rare variants and might be pathogenic, a detailed cose- In this cohort, most variants identified in the Nav1.4 gragation analysis and functional studies are still required channel were distributed in the S4-S6 transmembrane re- to valid the results. gions where the voltage sensors and pore formers are lo- Nine variants identified in Kir2.1 were located in the cated. A Novel variant p.Val240Met was located in the C-terminal intracytoplasmic tails. There is no hotspot variant cytoplasmic Domain I S4-S5 loop. The linker help con- in KCNJ2 identified in this Chinese cohort. These regions nected the voltage sensing domain S4 and pore-forming are considered to play an important role in maintaining nor- domains S5-S6 [23]. At normal conditions, depolarization mal function; thus, these mutations likely disrupt the ion Luo et al. BMC Neurology (2019) 19:92 Page 6 of 8

channels. Of these mutations, p.Pro186Thr, p.Arg189Ser, Using this NGS panel, we possibly expanded the p.Gly215Asp and p.Arg218Trp are located at sites in which spectrum of genotypes associated with PP (Fig. 4). NGS Kir2.1 and phosphatidylinositol-4,5-bisphosphate (PIP2) is not a perfect tool but is a method under development combine, which likely abolishes the direct and specific activa- for discovering new mutations that may be involved in tion of Kir2.1 [24]. Two highly frequent missense variants, disease conditions. By including primary myopathy/ i.e.,p.Arg528His and p.Arg1239His in CACNA1S gene were muscle dystrophy genes in the panel, we increased the observed in two patients. According to previous reports, pa- diagnostic yield from 61.67% (37/60) to 65.0% (39/60) in tients with these two mutations have a late onset age of 17.2 screening primary PP patients. But for some rare vari- ± 4.0 and 15.8 ± 8.8 years compared with that of other ants like RYR1 variants, the functional analysis is re- HypoPP cases [25, 26]. quired for further confirmation of the causality. RYR1 gene has originally been reported to be respon- Still, we were unable to achieve a definite diagnosis in sible for and malignant hyperther- the remaining 21(35.0%) cases. The relatively low depth mia. Two missense mutations of the RYR1 gene were and coverage in the GC-rich sequences of certain genes, demonstrated in two independent cases with PP in our which are likely causal CNV variations, and unknown cohort. The first mutation p.Glu2764Lys was previously genes may contribute to major causes of the found in a patient with [27]. panel-negative cases. Hopefully, solving the phenotype Another mutation p.Ala4143Val was novel with a positive/genotype negative mystery cases will shed new Polyphen-2score of 0.991 and a SIFT prediction score of light regarding novel pathogenic mechanisms underlying 0. This mutation is located in the Malignant the remaining cases of primary PP. hyperthermia domain III (exon 90–104). In a previous study, A homozygous RYR1 mutation (c.8816G > A Conclusions p.Arg2939Lys) in skeletal muscle was responsible for a Using targeted NGS, we achieved a diagnostic success 35-year-old male with congenital myopathy as well as a rate of 65% in a cohort of primary PP patients. We ex- typical periodic paralysis [3]. A discrepancy was demon- panded the spectrum of genotypes of primary PP and strated that the c.8816G > A nucleotide change was het- clinical phenotypes of known myopathy-related genes. erozygous at the genomic level. However, in our cases We’d like to attribute this diagnostic rate to the inclusive with RYR1 heterozygous mutations, it needs further ana- strategy of screening for other causal factors of hypokal- lysis with patient-derived skeletal muscle and cell models aemic muscle weakness and performing accurate clinical to establish the relationship between the RYR1 variants examinations and history inquiries prior to the interpret- and the phenotype. ation of the NGS results.

Fig. 4 The distribution of identified primary periodic paralysis related proteins Luo et al. BMC Neurology (2019) 19:92 Page 7 of 8

Additional files Author details 1Department of Neurology, Huashan Hospital, Fudan University, Shanghai 200040, China. 2Key Laboratory of Contraceptives and Devices, Shanghai Additional file 1: Table S1. Clinical features and genetic variants of the Institute of Planned Parenthood Research, institute of Reproduction and patients with primary periodic paralysis in this study. AD: autosomal development, Fudan University, Shanghai 200032, China. 3Department of dominant. AR: autosomal recessive. HypoPP: hypokalemic periodic clinical electrophysiology, Institute of Neurology, Huashan Hospital, Fudan paralysis. NormoPP: normokalemic periodic paralysis. Novel variants are in University, Shanghai 200040, China. 4State Key Laboratory of Genetic red. (DOCX 23 kb) Engineering, Department of Microbiology and Microbial Engineering, School Additional file 2: Table S2. Skeletal muscle ion channel genes with of Life Sciences, Fudan University, 200433, Shanghai, China. 5Department of GC-rich (> 70%) regions (DOCX 15 kb) Neurology, Jing’an District Center Hospital of Shanghai, Shanghai 200040, China.

Abbreviations Received: 28 June 2018 Accepted: 29 April 2019 ARVC: Arrhythmogenic right ventricular cardiomyopathy; BQSR: Base quality Score Recalibration; BWA: Burrows-Wheeler Aligner; CMAP: Compound muscle action potential; CNV: Copy number variant; CPEO: Chronic progressive external ophthalmoplegia; DHPR: Dihydropyridine receptor; References ER: Endoplasmic reticulum; ExAC: Exome Aggregation Consortium; 1. Suetterlin K, Männikkö R, Hanna MG. Muscle channelopathies: recent hg19: Human reference genome 19; HyperPP: Hyperkalemic periodic advances in genetics, pathophysiology and therapy. CurrOpin Neurol. 2014; paralysis; HypoPP: Hypokalemic periodic paralysis; NGS: Next generation 27(5):583–90. sequencing; NormoPP: Normokalemic periodic paralysis; OXPHOS: Oxidative 2. Marchant CL, Ellis FR, Halsall PJ, Hopkins PM, Robinson RL. Mutation analysis phosphorylation; PIP2: Phosphatidylinositol-4,5-bisphosphate; PP: periodic of two patients with hypokalemic periodic paralysis and suspected paralysis; SUNDS: Sudden unexplained nocturnal death syndrome; malignant hyperthermia. Muscle Nerve. 2004;30(1):114–7. WES: Whole exome sequencing; WGS: Whole genome sequencing 3. Zhou H, Lillis S, Loy RE, Ghassemi F, Rose MR, Norwood F, et al. NeuromusculDisord. 2010;20(3):166–73. Acknowledgements 4. Nigro V, Piluso G. Next generation sequencing (NGS) strategies for the We want to show our gratitude to the patients and the family members for genetic testing of myopathies. ActaMyol. 2012;31(3):196–200. their support in this study. We want to thank Amplicon gene company for 5. Dai Y, Wei X, Zhao Y, Ren H, Lan Z, Yang Y, et al. A comprehensive genetic providing extra support for the bioinformatic analysis. diagnosis of Chinese muscular dystrophy and congenital myopathy patients by targeted next-generation sequencing. NeuromusculDisord. 2015;25(8): – Funding 617 24. In this study, the sanger sequencing and bioinformatic analysis was 6. AnniEvilä MA, BjarneUdd PH. Targeted next-generation sequencing for sponsored by the National Natural Science Foundation of China Youth Fund detection of mutations in primary myopathies. NeuromusculDisord. 2016; (http://www.nsfc.gov.cn, NSFC_81701236 and NSFC_81870988). 26(1):7–15. 7. Song J, Luo S, Cheng X, Yue D, Zhu W, Lin J, et al. Clinical features and long exercise test in Chinese patients with Andersen-Tawil syndrome. Muscle Availability of data and materials Nerve. 2016;54(6):1059–63. The tabulated data will be available on request. 8. Todd EJ, Yau KS, Ong R, Slee J, McGillivray G, Barnett CP, et al. Next generation sequencing in a large cohort of patients presenting with Authors’ contributions before or at birth. Orphanet J Rare Dis. 2015;10:148. S.L. designed the study and mainly accomplished the manuscript draft. M.X., 9. Cox MP, Peterson DA, Biggs PJ. SolexaQA: at-a-glance quality X.N. and T.D. help in interpretation of the data. J.S., S.C., W.Z., L.Z., J.X., J.L. and assessment of Illumina second-generation sequencing data. BMC K.Q. contributed mainly in conception and design of the work. C.Z. revised bioinformatics. 2010;11:485. the manuscript. All authors read and approved the final manuscript. 10. Li H, Durbin R. Fast and accurate short read alignment with burrows- wheeler transform. Bioinformatics. 2009;25:1754–60. Ethical approval and consent to participant 11. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, et al. All procedures performed in studies involving human participants were in The genome analysis toolkit: a MapReduce framework for analyzing next- – accordance with the ethical standards of the institutional and/or national generation DNA sequencing data. Genome Res. 2010;20:1297 303. research committee and with the 1964 Helsinki declaration and its later 12. Wang K, Li M, Hakonarson H. ANNOVAR: functional annotation of amendments or comparable ethical standards. The study was approved by genetic variants from high-throughput sequencing data. Nucleic Acids the Institutional reviewing board of Huashan hospital Fudan University Res. 2010;38:e164. (2008–071). Each participant had signed an informed consent for the clinical 13. Stenson PD, Mort M, Ball EV, Shaw K, Phillips A, Cooper DN, et al. The data collection and genetic screening. For the participants who were under human gene mutation database: building a comprehensive mutation 18 years old when admitted into the study, the informed consent was repository for clinical and molecular genetics, diagnostic testing and – signed by the biological parents. The article does not contain any studies personalized genomic medicine. Hum Genet. 2014;133:1 9. with animals performed by any of the authors. 14. Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, et al. A method and server for predicting damaging missense mutations. Nat Methods. 2010;7:248–9. Consent to publication 15. Sim NL, Kumar P, Hu J, Henikoff S, Schneider G, Ng PC. SIFT web server: All the participants gave consent for publication of the potentially identifying predicting effects of amino acid substitutions on proteins. Nucleic Acids information including sex, gender, age and genetic screening results in the Res. 2012;40:W452–7. journal of BMC Neurology. 16. Plagnol V, Curtis J, Epstein M, et al. A robust model for read count data in exome sequencing experiments and implications for copy number variant Competing interests calling [J]. Bioinformatics. 2012;28(21):2747–54. The authors declare that they have no competing financial or non-financial 17. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and interests. guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015 Publisher’sNote May;17(5):405–24. Springer Nature remains neutral with regard to jurisdictional claims in 18. Poulin H, Gosselin-Badaroudine P, Vicart S, Habbout K, Sternberg D, published maps and institutional affiliations. Giuliano S, et al. Substitutions of the S4DIV R2 residue (R1451) in Nav1. Luo et al. BMC Neurology (2019) 19:92 Page 8 of 8

4 lead to complex forms of paramyotonia congenital and periodic paralyses. Sci Rep. 2018;8(1):2041. 19. Luo S, Sampedro Castañeda M, Matthews E, Sud R, Hanna MG, Sun J, et al. Hypokalaemic periodic paralysis and myotonia in a patient with homozygous mutation p.R1451L in Nav1.4. Sci Rep. 2018;8(1):9714. 20. Fouad G, Dalakas M, Servidei S, Mendell JR, Van den Bergh P, Angelini C, et al. Genotype-phenotype correlations of DHP receptor alpha 1-subunit gene mutations causing hypokalemic periodic paralysis. NeuromusculDisord. 1997;7(1):33–8. 21. Miller TM, Dias da Silva MR, Miller HA, Kwiecinski H, Mendell JR, Tawil R, et al. Correlating phenotype and genotype in the periodic paralysis. Neurology. 2004;63(9):1647–55. 22. Sternberg D, Maisonobe T, Jurkat-Rott K, Nicole S, Launay E, Chauveau D, et al. Hypokelaemic periodic paralysis type 2 caused by mutations at codon 672 in the muscle sodium channel gene SCN4A. Brain. 2001;124:1091–9. 23. Payandeh J, Scheuer T, Zheng N, Catterall WA. The crystal structure of a voltage-gated sodium channel. Nature. 2011;475(7356):353–8. 24. Plagnol V, Curtis J, Epstein M, Mok KY, Stebbings E, Grigoriadou S, et al. A robust model for read count data in exome sequencing experiments and implications for copy number variant calling. Bioinformatics. 2012;28:2747–54. 25. Wang Q, Liu M, Xu C, Tang Z, Liao Y, Du R, et al. Novel CACNA1S mutation causes autosomal dominant hypokalemic paralysis in a Chinese family. J Mol Med (Berl). 2005;83:203–8. 26. Houinato D, Laelye A, Adjien C, Adjagba M, Sternberg D, Hilbert P, et al. Hypokalaemic periodic paralysis due to the CACNA1S mutation in a large African family. NeuromusculDisord. 2007;17:419–22. 27. D’Avanzo N, Cheng WW, Doyle DA, Nichols CG. Direct and specific activation of human inward rectifier K+ channels by membrane phosphatidylinositol 4,5-bisphosphate. J Biol Chem. 2010;285:37129–32.