Targeted gene panel and genotype-phenotype correlation in children with developmental and epileptic encephalopathy

Epilepsy Research 141 (2018) 48–55

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Targeted gene panel and genotype-phenotype correlation in children with developmental and epileptic encephalopathy

T

⁎

Ara Koa, Song Ee Youna, Se Hee Kima, Joon Soo Leea, Sangwoo Kimb, Jong Rak Choic, , ⁎ ⁎ ⁎ Heung Dong Kima, , Seung-Tae Leec, , Hoon-Chul Kanga, a

Division of Pediatric Neurology, Epilepsy Research Institute, Severance Children’s Hospital, Department of Pediatrics, Yonsei University College of Medicine, 03722 Yonsei-ro 50-1, Seodaemun-gu, Seoul, Republic of Korea b Severance Biomedical Science Institute, Yonsei University College of Medicine, 03722 Yonsei-ro 50-1, Seodaemun-gu, Seoul, Republic of Korea c Department of Laboratory Medicine, Severance Hospital, Yonsei University College of Medicine, 03722 Yonsei-ro 50-1, Seodaemun-gu, Seoul, Republic of Korea

A R T I C L E I N F O

A B S T R A C T

Keywords: Developmental and epileptic encephalopathy Next-generation sequencing Gene panel

Objective: We performed targeted gene-panel sequencing for children with developmental and epileptic encephalopathy (DEE) and evaluated the clinical implications of genotype–phenotype correlations. Methods: We assessed 278 children with DEE using a customized gene panel that included 172 genes, and extensively reviewed their clinical characteristics, including therapeutic efficacy, according to genotype. Results: In 103 (37.1%) of the 278 patients with DEE, 35 different disease-causing monogenic mutations were identified. The diagnostic yield was higher among patients who were younger at seizure onset, especially those whose seizures started during the neonatal period, and in patients with drug-resistant epilepsy. According to epilepsy syndromes, the diagnostic yield was the highest among patients with West syndrome (WS) with a history of neonatal seizures and mutations in KCNQ2 and STXBP1 were most frequently identified. On the basis of genotypes, we evaluated the clinical progression and seizure outcomes with specific therapeutic regimens; these were similar to those reported previously. In particular, sodium channel blockers were effective in patients with mutations in KCNQ2 and SCN2A in infancy, as well as SCN8A, and interestingly, the ketogenic diet also showed diverse efficacy for patients with SCN1A, CDKL5, KCNQ2, STXBP1, and SCN2A mutations. Unfortunately, quinidine was not effective in 2 patients with migrating focal epilepsy in infancy related to KCNT1 mutations. Conclusion: Targeted gene-panel sequencing is a useful diagnostic tool for DEE in children, and genotype–phenotype correlations are helpful in anticipating the clinical progression and treatment efficacy among these patients.

1. Introduction Epileptic encephalopathy refers to a group of severe pediatric epilepsies in which epileptic activities contribute to cognitive delay or regression. (Scheffer et al., 2017) Recently, developmental and epileptic encephalopathy (DEE) was introduced as a new concept, because cognitive deterioration can also derive from genetic etiologies, irrespective of epileptic activity. (Scheffer et al., 2017) With advances in sequencing methods, monogenic mutations responsible for DEE have been identified, suggesting underlying genetic etiologies in DEE patients who were previously included in the unknown etiology group.

(Moller et al., 2015) Advanced sequencing methods have shortened the diagnostic process, and potential benefits from gene-based determination of clinical progression and therapeutic regimens might be expected in this era of emerging precision medicine. Therefore, here, we sought to elaborate our single-center experience of using targeted gene-panel sequencing to diagnose the genetic etiology of DEE and to reveal the clinical implications of gene-panel studies by extensively reviewing genotype–phenotype correlations in such patients.

Abbreviations: DEE, developmental and epileptic encephalopathy; EEG, electroencephalography; EIMFS, epilepsy of infancy with migrating focal seizures; EMAS, epilepsy with myoclonic atonic seizures; ID, intellectual disability; IQR, interquartile range; KD, ketogenic diet; LGS, lennox-gastaut syndrome; WS, west syndrome; EOEE, early-onset epileptic encephalopathy ⁎ Corresponding authors. E-mail addresses: [email protected] (J.R. Choi), [email protected] (H.D. Kim), [email protected] (S.-T. Lee), [email protected] (H.-C. Kang). https://doi.org/10.1016/j.eplepsyres.2018.02.003 Received 24 November 2017; Received in revised form 20 January 2018; Accepted 7 February 2018 Available online 12 February 2018 0920-1211/ © 2018 Elsevier B.V. All rights reserved.

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Table 1 Identified pathogenic mutations (n = 103, 35 genes). Pathogenic gene

n (%)

Pathogenic gene

n (%)

Pathogenic gene

n (%)

Pathogenic gene

n (%)

ALDH7A1 ARX BRAT1 CACNA1A CACNB4 CASK CDKL5 CHD2 DNM1

2 1 3 1 1 1 9 8 2

EEF1A2 GNAO1 GRIN2A HCN1 IQSEC2 KANSL1 KCNA1 KCNB1 KCNQ2

2 1 1 1 1 1 1 2 7

KCNT1 MECP2 PCDH19 PRODH SCN1A SCN1B SCN2A SCN3A SCN8A

3 (2.9) 5 (4.9) 3 (2.9) 1 (1.0) 11 (10.7) 1 (1.0) 5 (4.9) 1 (1.0) 5 (4.9)

SLC6A1 SLC9A6 STXBP1 SYN1 SYNGAP1 UBE3A WWOX ZEB2

3 2 7 1 5 2 1 2

(1.9) (1.0) (2.9) (1.0) (1.0) (1.0) (8.7) (7.8) (1.9)

(1.9) (1.0) (1.0) (1.0) (1.0) (1.0) (1.0) (1.9) (6.8)

(2.9) (1.9) (6.8) (1.0) (4.9) (1.9) (1.0) (1.9)

n, number.

2. Patients and methods

International League Against Epilepsy classification. (Berg et al., 2010) If the patient’s syndromic diagnoses changed over time, the first syndromic diagnosis was selected. Patients who developed seizures during the neonatal period and could not readily be categorized according to a syndrome but later developed West syndrome (WS) were grouped separately as “WS with neonatal seizures.” Drug-resistant epilepsy was defined as failure to achieve seizure freedom after adequate trials with 2 antiepileptic drugs. With regard to the efficacy of the therapeutic regimens, including diet therapy, we considered the regimens as effective if they contributed to more than 50% decrease in the seizure frequency from the baseline.

2.1. Patients A total of 280 unrelated pediatric patients with early-onset DEE of unknown etiology were recruited from the epilepsy clinic of Severance Children’s Hospital between March 2015 and June 2017. All patients met the following criteria: (1) seizure onset before the age of 3 years; (2) multiple epileptiform discharges with severely disorganized background activity on electroencephalography (EEG); (3) progressive developmental deterioration or a known developmental and epileptic encephalopathy syndrome; (4) no significant structural lesion detected on brain magnetic resonance imaging; (5) no metabolic abnormalities; and (6) no abnormalities detected on previous genetic tests. This study was approved by the Institutional Review Board of Severance Hospital.

2.4. Statistical analysis Data from statistical analyses are expressed as medians and interquartile ranges (IQRs) for continuous and ordinal variables, and as counts and percentages for categorical variables. The 2 groups were compared using the Chi-square test or Fisher’s exact tests for categorical and ordinal data, or the Mann–Whitney U-test for non-parametric continuous data. A p value of < 0.05 was considered significant. The Statistical Package for the Social Sciences (version 23.0; SPSS Inc., Chicago, IL, USA) was used for all analysis.

2.2. Targeted NGS gene panel A total of 172 genes known to be related to DEE were included in our gene panel; the genes are listed in Appendix A. Briefly, the processes for analyzing the sequencing data were as follows. Genomic DNA was extracted from the leukocytes of whole-blood samples using the QIAamp Blood DNA mini kit (Qiagen, Hilden, Germany). The pooled libraries were sequenced using a MiSeq sequencer (Illumina, San Diego, CA, USA) and the MiSeq Reagent Kit v2 (300 cycles). Sequencing data were aligned against appropriate reference sequences and analyzed using Sequencher 5.3 software (Gene Codes Corp., Ann Arbor, MI, USA). Parental studies were performed by Sanger sequencing on a 3730 DNA Analyzer with the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) if needed. Large exonic deletions and duplications were confirmed using the MLPA kit (MRC Holland). Classification of the variants followed a three-step approach: (1) conventional bioinformatics analysis, based on the nature of the mutation and frequencies in the normal population; (2) in silico analysis, with a literature review of the same variant and other variants in the same amino acid position; (3) a consensus discussion of genotype–phenotype correlations between geneticists and epileptologists, along with family studies and other confirmatory assays. Subsequently, the variants determined to be pathogenic or to be likely pathogenic on the basis of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology classification were considered as causative mutations for DEE. (Richards et al., 2015)

3. Results 3.1. Demographics and general characteristics Among the 278 patients (268 Koreans, 1 Mongolian, and 9 Caucasians), pathogenic monogenic mutations were identified in 103 (37.1%). Thirty-five different causative genes were found, with SCN1A being the most frequent (n = 11, 10.7%), followed by CDKL5 (n = 9, 8.7%), CHD2 (n = 8, 7.8%), KCNQ2 (n = 7, 6.8%), STXBP1 (n = 7, 6.8%), SCN2A (n = 5, 4.9%), SCN8A (n = 5, 4.9%), SYNGAP1 (n = 5, 4.9%), and others (Table 1). Thirty-four (33.0%) of 103 patients with identified mutations carried variants that had not been previously reported, which were therefore confirmed to be de novo mutations by trio sequencing. All pathogenic variants detected in this study are listed in the Appendix B. When the 103 patients with identified mutations were compared with the 175 patients who showed negative findings in the gene panel screen, the age of seizure onset was significantly earlier (p = 0.039) and the proportion of patients showing drug-resistant epilepsy was significantly larger (p < 0.001) among patients with identified mutations (Table 2). After controlling with age at seizure onset, presence of drugresistant epilepsy, sex, and epilepsy syndrome, age of seizure onset (OR 0.977, 95% CI 0.957–0.996, p = 0.019) and presence of drug-resistant epilepsy (OR 3.036, 95% CI 1.544–5.970, p = 0.001) were still significant predictors of achieving genetic diagnosis with gene panel study. The diagnostic yield was highest in patients with seizure onset during the neonatal period, with 80.0% (20 of 25) of patients proven to have causative mutations. This was significantly higher when compared

2.3. Clinical characteristics We reviewed the clinical features of DEE patients investigated using targeted gene-panel sequencing. The clinical characteristics included demographic profiles, classification of epilepsy syndromes, and seizure outcomes after therapeutic regimens and diet therapy. For epilepsy syndromes, the patients were classified according to the 2010 49

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Table 2 Demographic characteristics of patients with developmental and epileptic encephalopathy and comparison between patients with positive and negative results from targeted gene-panel sequencing.

Age at seizure onset (months) Sex (male) Drug-resistant epilepsy Prior genetic tests

Total (n = 278)

Identified pathogenic mutations (n = 103)

Negative results (n = 175)

p

7 (3–18) 155 (55.8%) 161 (57.9%) 107 (38.5%)

6 (2–18) 51 (49.5%) 74 (71.8%) 43 (41.7%)

7 (4–19) 104 (59.4%) 87 (49.7%) 64 (36.6%)

0.039 0.108 < 0.001 0.392

Data are presented as median (interquartile range) or number (percentage).

Lennox-Gastaut syndrome (LGS), SYNGAP1; for epilepsy with myoclonic atonic seizures (EMAS), SLC6A1; for unspecified generalized epilepsy, CDH2; and for unspecified focal epilepsy, PCDH19. Age of seizure onset for each genotype was relatively consistent, and the median onset age of seizures was 3 days in patients with KCNQ2-related encephalopathy; 7 days in STXBP1-related conditions; 1 month in KCNT1-related conditions; 3 months in CDKL5-, SCN8A-, and BRAT1related conditions; and 6 months in SCN1A-related conditions. The age at seizure onset in patients with CHD2 and SYNGAP1 encephalopathy was relatively high, with a median seizure onset age of 19 and 26 months, respectively (Appendix D). The details of the clinical characteristics, including seizure outcomes according to therapeutic regimens, are described below.

to the patients whose seizures began after 30 days of age who shows a diagnostic yield of 32.8% (p < 0.001). The proportions of identified gene mutations decreased as the age at seizure onset increased (Appendix C).

3.2. Genotype–phenotype correlations The 278 patients could be classified into 10 groups according to the epilepsy syndromes; 119 (42.8%) were classified as showing WS. The diagnostic yield of the gene-panel study differed significantly among patients with the different epilepsy syndromes (p < 0.001,), with the highest diagnostic yield in patients with WS with neonatal seizures (100.0%), followed by those with Ohtahara syndrome (85.7%), and others (Fig. 1). Fig. 2 shows the range of ages at seizure onset in our patients and the mutations identified in each epilepsy syndrome. For patients with WS with neonatal seizures, KCNQ2 and STXBP1 were the most frequently identified disease-causing genes; for those with Ohtahara syndrome, KCNQ2; for epilepsy of infancy with migrating focal seizures (EIMFS), KCNT1; for WS, CDKL5; for Dravet syndrome, SCN1A; for

3.3. SCN1A Eleven patients were identified with mutations in SCN1A (sodium channel, neuronal type 1, alpha subunit gene). A summary of the clinical characteristics are shown in Table 3. All patients showed phenotypes that were concordant with Dravet syndrome. These 11 patients

Fig. 1. Positivity rate of targeted gene-panel sequencing for developmental and epileptic encephalopathy according to epilepsy syndromes. (WS, West syndrome; EIMFS, epilepsy of infancy with migrating focal seizures; GE, generalized epilepsy; EMAS, epilepsy with myoclonic atonic seizures; LGS, Lennox-Gastaut syndrome; LKS, Landau-Kleffner syndrome)

50

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Fig. 2. Genotype-phenotype correlations. The bar indicates the range of seizure onset age (months) observed in our cohort for each syndrome, and genes identified to have disease-causing mutations for each syndrome are listed in an order of decreasing frequency. The numbers before the genes indicate the identified frequency for each gene. (WS, West syndrome; EIMFS, epilepsy of infancy with migrating focal seizures; LGS, Lennox-Gastaut syndrome; EMAS, epilepsy with myoclonic atonic seizures; GE, generalized epilepsy; FE, focal epilepsy)

3.5. CHD2

comprised 61.1% of those with total Dravet syndrome (n = 18), with another 6 patients without identified pathogenic variants and 1 with a mutation in SCN1B.

Eight patients showed mutations in CHD2 (chromodomain helicase DNA-binding protein 2 gene). All patients presented predominantly with myoclonic seizures, and clinical photosensitivity was observed in 5 patients (62.5%). The EEGs of all these patients showed generalized epileptiform discharges, and 2 patients were diagnosed with EMAS, 1 with LGS, and 5 were classified as having unspecified generalized epilepsy. All patients showed normal development until seizure onset, had their first seizures during childhood at a median age of 19 months, and showed developmental impairment after seizure onset. Five patients, with disease duration of less than 10 years, currently show mild ID with relatively near normal social quotients on the social maturity scale. However, 2 patients with longer follow-up periods have shown continued developmental regression to severe ID. In 6 (75.0%) patients, valproate was the most effective medication, while 2 (25.0%) patients have drug-resistant epilepsy.

3.4. CDKL5 Nine patients had mutations in the CDKL5 (cyclin-dependent kinaselike 5 gene) in our study. Five patients presented with tonic seizures first, and shortly afterwards developed spasms and later showed hypsarrhythmia on EEG and were diagnosed with WS. Two patients presented with spasms with hypsarrhythmia on EEG, and were also diagnosed with WS. The remaining 2 patients who had an earlier seizure onset age (15 and 50 days, respectively) presented with tonic seizures with a burst-suppression pattern on EEG, and were diagnosed with Ohatahara syndrome; the condition in both evolved into WS at the age of 3 months. All these patients showed early developmental delay before seizure onset, and showed profound intellectual disability (ID); 7 (77.8%) were unable to make eye contact or control their heads. All patients also showed drug-resistant epilepsy, and a ketogenic diet (KD) was attempted in all 9 patients, but was effective in only 1 patient. In 2 patients who were followed up for more than 10 years, seizures evolved into LGS. Corpus callosotomy was performed in both of them, which resulted in 50% and 75% seizure reduction after the surgery, respectively.

3.6. KCNQ2 Seven patients were found to harbor mutations in KCNQ2 (potassium channel, voltage-gated, KQT-like subfamily, member 2 gene). Patients with KCNQ2 encephalopathy showed the earliest seizure onset, and all patients had seizure onset within the first 9 days of life. All patients presented with tonic seizures, but EEGs of 4 patients showed a burst-suppression pattern, while the other 3 patients showed focal epileptiform discharges. The former 4 patients were diagnosed with 51

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KD (4); Phenytoin (2); Oxcarbazepine (2) KD (4); Vigabatrin (1); Prednisolone (1)

KD (3); Phenytoin (1); Oxcarbazepine (1)

Phenytoin (2); Carbamazepine (1) Valproic acid (1); Clobazam (1); Zonisamide (1)

Mild to severe ID

Profound ID Mild to profound ID

Mild to profound ID

Moderate to profound ID Moderate to severe ID

M (in all patients), T, AA T T, C, S, BA

S, T, A, BA

S, T M, A, AA

5:3

3:2

2:3 3:2

4:3 5:2

19 (IQR 17–21, range 12–48) months

3 (IQR 2–4, range 2–9) days 7 days (IQR 7 days to 2 months, range 3 days to 7 months) 5 months (IQR 2.5–33 months, range 1 day to 36 months) 3 (IQR 1.5–5, range 0.5–5) months 26 (IQR 23–34, range 22–36) months

GE, unspecified (5); LGS (2); Doose (1)

WS with neonatal seizure (3); Ohtahara (4) WS with neonatal seizure (3); Ohtahara (2); WS (1); FE, unspecified (1) WS (2); Ohtahara (1); LGS (1); FE, unspecified (1)

8

7 7

5

5 5

CHD2

KCNQ2 STXBP1

SCN2A

SCN8A SYNGAP1

WS (4); WS with neonatal seizure (1) LGS (3); Doose (1); FE, unspecified (1)

6:5 2:7 6 (IQR 5–8, range 2–12) months 3 (IQR 2–5, range 0.5–10) months All Dravet syndrome WS (7); Ohtahara (2) 11 9 SCN1A CDKL5

n, number; sz, seizure; DS, Dravet symdrome; IQR, interquartile range; M, myoclonic; T, tonic; TC, tonic-clonic; FC, focal cognitive; ID, intellectual disability; KD, ketogenic diet; WS, West syndrome; S, spasms; GE, generalized epilepsy; LGS, LennoxGastaut syndrome; AA, atypical absence; BA, behavioral arrest; C, clonic; A, atonic.

KD (6); Stiripentol (3) Prednisolone (2); Callosotomy (2); Topiramate (1); KD (1) Valproate (6); Ethosuximide (1) Mild to severe ID Profound ID M, T, TC S (T shortly before), T

Sex (M:F) Seizure onset Syndromes n Pathogenic genes

Table 3 Clinical characteristics of the eight most frequently identified genotypes.

Most effective treatment for seizure control Development Initial seizure types

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Ohtahara syndrome, and the condition in all 7 patients later evolved into WS. All patients showed profound ID, with none showing any signs of development. All patients had drug-resistant epilepsy, but the KD had favorable responses in 6 patients. Sodium channel blockers were administered in 4 patients and these were effective in controlling their seizures. 3.7. STXBP1 Seven patients had mutations in STXBP1 (syntaxin-binding protein 1 gene). The median seizure onset age was 7 days, which was relatively early. Five patients presented with focal or generalized seizures during the neonatal period, 2 were diagnosed with Ohtahara syndrome with a burst-suppression pattern on EEG, and 3 showed focal epileptiform discharge. The remaining 2 patients with a later seizure onset age, at 2 and 8 months, presented with spasms, hypsarrhythmia on EEG, and were diagnosed with WS. The condition in the former 5 patients also evolved into WS. Seizures were drug resistant in half of the patients, and various degrees (mild to severe) of ID were observed. Ketogenic diet was effective in seizure reduction in all 4 patients who tried the dietary therapy. 3.8. SCN2A Five patients had pathogenic variants in SCN2A (sodium channel, neuronal type 2, alpha subunit gene). The age of seizure onset had a bimodal distribution; 3 patients had seizure onset at 1 day, 2 patients at 5 months, and 2 patients at 30 and 36 months each. The first 3 patients presented with Ohtahara syndrome and WS, which later evolved into LGS with a significant developmental impact (3 were unable to make eye contact or control their heads). Two patients with seizure onset at 1 day and 5 months, respectively, were given sodium channel blockers and showed significant seizure reduction. One patient with seizure onset age of 5 months was not tried with sodium channel blockers. The other 2 patients with later seizure onset age of 30 and 36 months were both diagnosed with LGS and unspecified focal epilepsy. Both patients showed normal development before seizure onset, and regressed thereafter, currently showing mild ID. Sodium channel blockers were administered to both patients without effect and were discontinued. 3.9. SCN8A Five patients were identified with pathogenic variants in SCN8A (sodium channel, neuronal type 8, alpha subunit gene). One patient presented with neonatal seizures that later developed into WS, and the other 4 all presented with WS. Later, the condition in all 5 patients evolved into LGS, and the patients showed significant developmental delay. Three patients (60.0%) had drug-resistant epilepsy; sodium channel blockers were administered to 4 patients, with a favorable response in 3 (75.0%) patients. 3.10. SYNGAP1 Five patients had disease-causing variants in SYNGAP1 (synaptic Ras-GTPase-activating protein 1 gene). The median seizure onset age was 26 months, which was relatively late. All patients had generalized seizures, such as myoclonic, atonic, or atypical absence seizures, with EEGs showing predominantly generalized epileptiform discharges. Four patients were diagnosed with LGS and 1 with EMAS. In 1 (20.0%) patient, seizures were intractable to medication. All patients showed developmental delay prior to seizure onset, and currently show severe ID, except for 1 patient with moderate ID. 4. Discussion We defined 35 different disease-causing monogenic mutations in 52

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rearrangements; localization of the mutations in the protein; the loss- or gain-of-function mechanisms caused by the mutations; epigenetic factors; and modifier genes. (Gennaro et al., 2006; Miceli et al., 2013; Nakamura et al., 2013; Seltzer et al., 2014; Singh et al., 2009; Syrbe et al., 2015) However, patients with the various pathogenic genes do share some common features, such as the temporal expression of symptoms and the type of epilepsy syndromes, as described above. This was more evident in some cases, such as in those with KCNT1 mutations, where all 3 patients presented with EIMFS, and as in Dravet syndrome, where patients almost exclusively had SCN1A as the causative gene (91.7% of genetically diagnosed patients). The ultimate goal of genetic diagnosis is targeted therapy. However, available therapies targeted to known genetic mutations are still limited to a few genes, such as KD for SCL2A1, retigabine for KCNQ2, memantine for GRIN2A or GRIN2B, and quinidine for KCNT1. (Gunthorpe et al., 2012; Kass et al., 2016; Milligan et al., 2014; Pierson et al., 2014; Platzer et al., 2017) Three EIMFS patients in our cohort who had pathogenic variants in KCNT1 showed intractable seizures, and 2 of these were administered quinidine; this was discontinued for 1 patient before the therapeutic level was reached due to QT prolongation, while another patient, in whom the therapeutic serum concentration was reached, did not show any effects in seizure reduction. Therefore, more studies are warranted for achieving targeted therapy, including reprogramming of stem cells or gene therapy, based on molecular diagnoses. In other cases, patients with each genetic mutation in this study showed clinical characteristics that were similar to those described previously, and also similar responses to specific therapies. Besides retigabine, sodium channel blockers are also known to be effective in seizure control in KCNQ2 encephalopathy patients, probably through modulation of the sodium channel that affects the function of the channel complex including the potassium channel. (Nguyen et al., 2012; Pisano et al., 2015) In this study, sodium channel blockers were administered to 4 patients, and were effective but did not completely control the seizures in all 4 patients. Wolff et al. reported that mutations in SCN2A cause 2 distinct phenotypes: early infantile onset (< 3 months) and infantile/childhood onset (≥3 months) encephalopathies. (Wolff et al., 2017) The early infantile form was associated with gain-of-function mutations, and therefore showed good response to sodium channel blockers, while the later onset form was associated with a loss-of function and sodium channel blockers were rarely effective or sometimes worsened the seizures. (Wolff et al., 2017) Also, early infantile form were all identified with missense mutations, while later onset form was associated with both missense and nonsense mutations, and patients with nonsense mutations all had seizure onset beyond the first year of life. In our study, the patients could be distinguished into earlier and later onset groups; earlier onset patients all had missense mutations and showed a good response to sodium channel blockers, while they were not effective in later onset patients with nonsense mutations, resulting in discontinuation of the medication. For mutations in SCN8A, most functional analyses to date have revealed gain-of-function effects, but some variants also produced loss-of-function effects in vitro. (Blanchard et al., 2015) In our study, the majority (75.0%) of the patients showed a good response to sodium channel blockers.

37.1% patients with DEE. These patients had a lower age at seizure onset and had more intractable seizures than patients in whom mutations were not identified with the gene panel screening. With the advent of sequencing methods that enable sequencing of several DNA regions in a single reaction, there have been significant advances in the identification of epilepsy-related genes. (Moller et al., 2015) Monogenic epilepsy, in which a single variant with a large effect is considered causative, is far less common than complex genetic epilepsy, in which a combinatorial effect of multiple variants is considered causative. (Mei et al., 2017) However, previous studies with NGS gene panels for DEE have shown substantial diagnostic yields of about 20–40%, which is not inferior to the diagnostic yield of whole exome sequencing. (Allen et al., 2013; Carvill et al., 2013; de Kovel et al., 2016; Gokben et al., 2017; Mercimek-Mahmutoglu et al., 2015; Michaud et al., 2014; Ortega-Moreno et al., 2017; Parrini et al., 2017; Segal et al., 2016; Trump et al., 2016; Zhang et al., 2017) Some of recent studies include a study with 175 Chinese patients with earlyonset epileptic encephalopathy investigated with gene panel comprising 17 genes which identified disease-causing variants in 32% of patients, a study with 87 patients with epilepsy and developmental delay investigated with 106 genes gene panel in which 19.5% of patients were identified with pathogenic variants, a study with 349 patients with drug-resistant epilepsy and seizure onset before 1 year of age who were investigated with 95-genes gene panel and showed 26.6% of diagnostic yield, and a study with 400 patients with early-onset seizures and severe developmental delay who were investigated with 46-genes gene panel and showed 18% of diagnostic yield. (Ortega-Moreno et al., 2017; Parrini et al., 2017; Trump et al., 2016; Zhang et al., 2017) Direct comparisons between studies for factors influencing diagnostic yields are difficult as cohorts and genes included in the panels are different, but slightly higher diagnostic yield in our study may be attributable to higher availability of parental samples (90.1% of 131 patients whose parental samples were needed to investigate variants with unknown significance) to confirm de novo occurrence of variants. In 41.7% of patients in whom disease-causing variants were identified in this study, results from previous genetic tests prior to the gene-panel study were uninformative. Therefore, monogenic variants, especially de novo variants, have an important role in DEE, and at present, targeted genepanel sequencing is the most cost-effective diagnostic option for epilepsy patients with suspected genetic etiology. (Helbig et al., 2016; Mei et al., 2017) Here, the proportion of patients with drug-resistant epilepsy was significantly higher among patients with identified mutations, which is concordant with the results of previous reports that showed higher diagnostic yields in cohorts with severe drug-resistant epilepsy. (Mei et al., 2017) These observations have not been explained to date; however, possible reasons are that patients with somatic mutations occurring only in the brain, those with a low rate of mosaic mutations, or patients with complex genetic epilepsy obtaining negative results in gene panels, may have less severe symptoms than patients carrying monogenic mutations with large effects. The seizure onset age was significantly lower in patients with identified mutations. This observation is also in accord with those of previous studies, which showed that seizures with an earlier age at onset resulted in higher molecular yields. (Helbig et al., 2016; Parrini et al., 2017) The timing of seizure onset for each pathogenic gene was relatively consistent among patients, as seizures started at the age at which the expression of a gene with the pathogenic mutation is required for physiological neuronal development. (McTague et al., 2016) Therefore, patients with pathogenic monogenic variants with strong effects may develop genetic dysfunctions earlier than patients with presumably somatic mosaicism or a polygenic disease basis. Naturally, the diagnostic yields were also higher in catastrophic epilepsy syndromes with earlier seizure onset, such as WS with neonatal seizure, Ohtahara syndrome, and EIMFS. Factors accounting for phenotypic pleiotropy can include the type and timing of mutations, including somatic mosaicism or genomic

5. Conclusion Monogenic mutations, especially de novo monogenic variants, are an important underlying etiology for DEE, and targeted gene-panel sequencing is an effective diagnostic tool for DEE. The diagnostic yield is higher in drug-resistant epilepsy, and in patients with earlier seizure onset especially during the neonatal period. Although phenotypic pleiotropy exists, we could confirm correlation of genotypes with the clinical progress and seizure outcomes to specific therapeutic regimens that were similar to those described in previous studies. 53

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This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI15C1601) and by a faculty research grant of Yonsei University College of Medicine for 2013 (6-2013-0031) and 2015(6-2015-0140). Conflict of interest The authors have no conflicts of interest relevant to this article to disclose. Acknowledgements None. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.eplepsyres.2018.02.003. References Allen, A.S., Berkovic, S.F., Cossette, P., Delanty, N., Dlugos, D., Eichler, E.E., Epstein, M.P., Glauser, T., Goldstein, D.B., Han, Y., Heinzen, E.L., Hitomi, Y., Howell, K.B., Johnson, M.R., Kuzniecky, R., Lowenstein, D.H., Lu, Y.F., Madou, M.R., Marson, A.G., Mefford, H.C., Esmaeeli Nieh, S., O'Brien, T.J., Ottman, R., Petrovski, S., Poduri, A., Ruzzo, E.K., Scheffer, I.E., Sherr, E.H., Yuskaitis, C.J., Abou-Khalil, B., Alldredge, B.K., Bautista, J.F., Berkovic, S.F., Boro, A., Cascino, G.D., Consalvo, D., Crumrine, P., Devinsky, O., Dlugos, D., Epstein, M.P., Fiol, M., Fountain, N.B., French, J., Friedman, D., Geller, E.B., Glauser, T., Glynn, S., Haut, S.R., Hayward, J., Helmers, S.L., Joshi, S., Kanner, A., Kirsch, H.E., Knowlton, R.C., Kossoff, E.H., Kuperman, R., Kuzniecky, R., Lowenstein, D.H., McGuire, S.M., Motika, P.V., Novotny, E.J., Ottman, R., Paolicchi, J.M., Parent, J.M., Park, K., Poduri, A., Scheffer, I.E., Shellhaas, R.A., Sherr, E.H., Shih, J.J., Singh, R., Sirven, J., Smith, M.C., Sullivan, J., Lin Thio, L., Venkat, A., Vining, E.P., Von Allmen, G.K., Weisenberg, J.L., Widdess-Walsh, P., Winawer, M.R., 2013. De novo mutations in epileptic encephalopathies. Nature 501, 217–221. Berg, A.T., Berkovic, S.F., Brodie, M.J., Buchhalter, J., Cross, J.H., van Emde Boas, W., Engel, J., French, J., Glauser, T.A., Mathern, G.W., Moshe, S.L., Nordli, D., Plouin, P., Scheffer, I.E., 2010. Revised terminology and concepts for organization of seizures and epilepsies: report of the ILAE Commission on Classification and Terminology, 2005–2009. Epilepsia 51, 676–685. Blanchard, M.G., Willemsen, M.H., Walker, J.B., Dib-Hajj, S.D., Waxman, S.G., Jongmans, M.C., Kleefstra, T., van de Warrenburg, B.P., Praamstra, P., Nicolai, J., Yntema, H.G., Bindels, R.J., Meisler, M.H., Kamsteeg, E.J., 2015. De novo gain-of-function and lossof-function mutations of SCN8A in patients with intellectual disabilities and epilepsy. J. Med. Genet. 52, 330–337. Carvill, G.L., Heavin, S.B., Yendle, S.C., McMahon, J.M., O'Roak, B.J., Cook, J., Khan, A., Dorschner, M.O., Weaver, M., Calvert, S., Malone, S., Wallace, G., Stanley, T., Bye, A.M., Bleasel, A., Howell, K.B., Kivity, S., Mackay, M.T., Rodriguez-Casero, V., Webster, R., Korczyn, A., Afawi, Z., Zelnick, N., Lerman-Sagie, T., Lev, D., Moller, R.S., Gill, D., Andrade, D.M., Freeman, J.L., Sadleir, L.G., Shendure, J., Berkovic, S.F., Scheffer, I.E., Mefford, H.C., 2013. Targeted resequencing in epileptic encephalopathies identifies de novo mutations in CHD2 and SYNGAP1. Nature Genet. 45, 825–830. Gennaro, E., Santorelli, F.M., Bertini, E., Buti, D., Gaggero, R., Gobbi, G., Lini, M., Granata, T., Freri, E., Parmeggiani, A., Striano, P., Veggiotti, P., Cardinali, S., Bricarelli, F.D., Minetti, C., Zara, F., 2006. Somatic and germline mosaicisms in severe myoclonic epilepsy of infancy. Biochem. Biophys. Res. Commun. 341, 489–493. Gokben, S., Onay, H., Yilmaz, S., Atik, T., Serdaroglu, G., Tekin, H., Ozkinay, F., 2017. Targeted next generation sequencing: the diagnostic value in early-onset epileptic encephalopathy. Acta Neurol. Belg. 117, 131–138. Gunthorpe, M.J., Large, C.H., Sankar, R., 2012. The mechanism of action of retigabine (ezogabine), a first-in-class K+ channel opener for the treatment of epilepsy. Epilepsia 53, 412–424. Helbig, K.L., Farwell Hagman, K.D., Shinde, D.N., Mroske, C., Powis, Z., Li, S., Tang, S., Helbig, I., 2016. Diagnostic exome sequencing provides a molecular diagnosis for a significant proportion of patients with epilepsy. Genet. Med. 18, 898–905. Kass, H.R., Winesett, S.P., Bessone, S.K., Turner, Z., Kossoff, E.H., 2016. Use of dietary therapies amongst patients with GLUT1 deficiency syndrome. Seizure 35, 83–87. McTague, A., Howell, K.B., Cross, J.H., Kurian, M.A., Scheffer, I.E., 2016. The genetic landscape of the epileptic encephalopathies of infancy and childhood. Lancet Neurol. 15, 304–316. Mei, D., Parrini, E., Marini, C., Guerrini, R., 2017. The impact of next-generation sequencing on the diagnosis and treatment of epilepsy in paediatric patients. Mol.

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