Epilepsy & Behavior 44 (2015) 61–66

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Predictive value of EFHC1 variants for the long-term seizure outcome in juvenile myoclonic epilepsy Felix von Podewils a,⁎,1, Victoria Kowoll a,1, Winnie Schroeder b, Julia Geithner a,c, Zhong I. Wang d, Bernadette Gaida a, Paula Bombach a, Christof Kessler a, Ute Felbor b, Uwe Runge a a

Department of Neurology, Epilepsy Center, University of Greifswald, Greifswald, Germany Department of Human Genetics, University Medicine Greifswald and Interfaculty Institute of Genetics and Functional Genomics, Ernst Moritz Arndt University, Greifswald, Germany Epilepsy Center Berlin-Brandenburg, Berlin, Germany d Epilepsy Center, Neurological Institute, Cleveland Clinic Foundation, Cleveland, OH, USA b c

a r t i c l e

i n f o

Article history: Received 16 October 2014 Revised 12 December 2014 Accepted 13 December 2014 Available online xxxx Keywords: Juvenile myoclonic epilepsy EFHC1 Genetic variants Outcome predictors

a b s t r a c t Objective: This study aimed to determine the contribution of EFHC1 variants to the phenotypic variability of juvenile myoclonic epilepsy (JME) and to evaluate their diagnostic value regarding previously identified clinical longterm seizure outcome predictors in a consecutive cohort of patients with JME. Methods: Thirty-eight probands and three family members affected with JME were studied at a tertiary epilepsy center with a review of their medical records and a subsequent face-to-face interview. All coding EFHC1 exons and adjacent exon/intron boundaries were directly sequenced. Results: The previously reported EFHC1 mutation F229L was found in two cases who presented with early generalized tonic–clonic seizure (GTCS) onset and appeared to be associated with milder subtypes of JME. Variant R294H was identified in two further probands who had a subtype of JME developing from childhood absence epilepsy. However, segregation of the phenotype with this variant could not be confirmed in one family. Conclusions: Our findings corroborate the heterogeneity of JME as an electroclinical epilepsy syndrome and provide evidence that genetic factors may influence and help predict the long-term seizure outcome in patients with JME. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Juvenile myoclonic epilepsy (JME), a specific electroclinical syndrome, is characterized by mandatory bilateral myoclonic seizures (BMSs) alone or combined with generalized tonic–clonic seizures (GTCSs) and/or absence seizures (ABSs) [1–8] as well as generalized spikes and polyspikes ≥3 Hz on interictal electroencephalography (EEG) [9,10]. Approximately 30% of patients with JME show photoparoxysmal responses (PPRs) [7,11–14]. Several recent long-term outcome studies on JME identified the occurrence of GTCSs preceded by BMSs, a long duration of unsuccessful treatment, antiepileptic drug (AED) polytherapy, and the additional manifestation of ABSs as predictors for a poor long-term outcome, whereas complete GTCS remission under AEDs increases the chance for complete seizure freedom [15–18]. Early seizure onset has been

⁎ Corresponding author at: University of Greifswald, Department of Neurology, Epilepsy Center, Sauerbruchstrasse, 17489 Greifswald, Germany. Tel.: +49 3834 866815; fax: +49 3834 866875. E-mail address: [email protected] (F. von Podewils). 1 These authors contributed equally to the manuscript.

http://dx.doi.org/10.1016/j.yebeh.2014.12.016 1525-5050/© 2014 Elsevier Inc. All rights reserved.

associated with seizure freedom without AEDs [17]; the occurrence of PPRs is predictive of seizure relapse after AED discontinuation [16]. Genetic linkage analyses identified about 15 loci that were linked to JME [19]. In addition to GABRA1 (alpha 1 subunit of the GABAA receptor) and CLCN2 (chloride channel 2 gene), heterozygous mutations in EFHC1 have been associated with JME [20] in 3 to 9% of consecutive families with JME. [21,22]. EF-hand domain containing 1 (EFHC1) encodes for the approximately 70 kDa protein myoclonin1 which contains three consecutive DM10 domains and an EF-hand calcium-binding motif at the C-terminus [20]. Efhc1 knockout mice showed spontaneous myoclonic seizures and increased seizure susceptibility to pentylenetetrazol that may well mimic the patients' epileptic phenotypes in both homozygous and heterozygous conditions, indicating a putative role of myoclonin1 deficiency in the genesis of epilepsies [23]. Despite the classical theories of channelopathies causing IGE, De Nijs et al. hypothesized JME to be a developmental disease and EFHC1 mutations to induce subtle malformation of cortical development that lead to abnormal epileptogenic circuitry [24,25]. The aims of this prospective study were to identify EFHC1 mutations and to determine their contribution to the phenotypic variability of JME. We also intended to evaluate their diagnostic value regarding previously

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identified clinical long-term seizure outcome predictors in a consecutive cohort of patients with JME.

3. Results 3.1. Patient demographics

2. Materials and methods 2.1. Patients The local Institutional Review Board approved this study. Written informed consent was obtained from all enrolled patients. The study was conducted within the referral area of a tertiary care epilepsy center (total population: ~500,000). Forty-one patients of Caucasian origin with JME were prospectively recruited from the inpatient and outpatient clinics of the Epilepsy Center Greifswald from January 2011 to January 2014. Inclusion criteria were as follows: (1) diagnosis of JME, (2) normal neurological examination and overall intelligence, (3) at least one routine EEG (international 10–20 system of electrode placement), and (4) normal clinical MRI. We excluded patients with severe brain trauma and a history of epilepsy syndromes other than JME and childhood absence epilepsy or juvenile absence epilepsy (CAE/ JAE) that converted into JME. Diagnosis of JME was made by experienced epileptologists (UR, FvP, and JG) on the basis of the patients' clinical history as well as the EEG including video-EEG monitoring in the majority of the patients. Photoparoxysmal responses were classified according to the classification of Waltz et al. [26]. Clinical data – such as seizure types, age at onset, detailed medical history, provoking factors including PPRs, and family history – were collected by reviewing the medical records and during a face-to-face interview. Seizure remission was defined as a terminal seizure-free period up to enrollment of at least five years. 2.2. Molecular analysis Genomic DNA was extracted from peripheral blood leukocytes by a salting-out method [27]; all coding EFHC1 exons and adjacent exon/intron boundary regions were directly sequenced on an ABI 3130xl automated sequencer (Applied Biosystems, Life Technologies GmbH, Darmstadt, Germany) with M13-tagged standard sequencing primers and analyzed using SeqPilot software (JSI medical systems GmbH, Kippenheim, Germany) (details of primers and reaction conditions are available on request). GenBank and Ensembl accession numbers for the EFHC1 gene are NM_018100.3 and ENST00000371068, respectively. Sequences were analyzed in SeqPilot with the Ensembl data set. Deoxyribonucleic acid mutation numbering is based on cDNA sequence, with +1 corresponding to the A of the ATG translation initiation codon. After assessing the Human Gene Mutation Database (HGMD® Professional) [28], in silico analyses were performed to evaluate the pathogenicity of exonic missense variants using the following prediction programs: MutationTaster (http://www.mutationtaster.org/) [29], PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) [30], and SIFT (http://sift.jcvi. org/) [31]. Minor allele frequencies (MAFs) were deduced from the Exome Variant Server (NHLBI GO Exome Sequencing Project (ESP), Seattle, WA; URL: http://evs.gs.washington.edu/EVS/ [10/2014]).

Details of all patients included in the study are given in Table 1. We enrolled 38 probands (25 females) and three family members affected with JME. The mean age of all included probands was 39.9 years (SD ± 16.91; range: 18–78); the mean age at epilepsy onset (first recognized seizure) was 13.6 years (SD ± 4.86; range: 2–27), and the mean duration of epilepsy was 26.2 years (SD ± 17.92; range: 3–67). The majority of patients had all three types of seizures typical for JME (BMSs, ABSs, and GTCSs) (56.1%); 12 (29.3%) patients had BMSs and GTCSs; 4 (9.8%) patients had BMSs and ABSs, and 2 (4.9%) patients had BMSs only. Mean number of AEDs tried up to the time of enrollment was 3.1 (SD ± 1.58; range: 1–7). Seventeen (41.5%) patients had PPRs, all of them were classified as either type III or type IV according to Waltz et al. [26]. Frequencies of outcome predictors are given in Table 2. 3.2. Genetics Among the 38 index patients analyzed in this study, the EFHC1 missense mutation c.685TN C (p.F229L) was found twice. F229L has been classified as a mutation occurring in two Mexican families with JME but none in 382 ethnically matched controls [20]. In Caucasians, F229L was found to have a minor allele frequency (MAF) of 0.47% (Exome Variant Server). Cosegregation has been described in several families with epilepsy [20,32]. MutationTaster and PolyPhen 2 suggest an influence on protein function which has been supported by functional studies demonstrating that F229L promotes calcium-induced apoptosis [20] and acts in a dominant-negative manner to impair mitotic spindle organization [24]. Variant c.881G NA (p.R294H) was identified in two further probands. R294H has a MAF of 1.05% (Exome Variant Server), resides within the second DM10 domain of the EFHC1 protein, and was predicted as disease-causing by Mutation Taster, as probably damaging by Polyphen2, and as damaging by SIFT. However, cosegregation with JME could not be confirmed in one of the families analyzed in the present study (Fig. 1). Sequence variant c.545GN A (p.R182H) was found in three further probands. R182H has been classified as polymorphism which is slightly enriched in disease cases [20,33] and was shown to cosegregate with JME in a family from Belize and in a family form Italy [20,32].

3.2.1. Associations of EFHC1 variants with JME phenotypes and outcome predictors Associations of F229L, R294H, and R182H variants in EFHC1 with JME phenotypes, the clinical outcome, and previously identified JME outcome predictors were investigated. The occurrence of these EFHC1 variants was associated with early onset GTCSs (≤ 12 years of age) (66.7% vs. 12.5%; p = 0.022b; OR: 13), a higher risk of status epilepticus (p = 0.001b), and a decreased risk of BMSs in series (p = 0.05b). Details of several important aspects of individual EFHC1 variants are described below and summarized in Table 3.

2.3. Statistics SPSS 22.0 (IBM Co., Armonk, NY, U.S.A.) was used for statistical processing of the data. The statistical methods were descriptive statistics with frequency analysis and cross-tab analysis, as well as mean and standard deviation (SD) calculation in parametric data. Sensitivity, specificity, and predictive values were calculated for each predictive parameter. Statistical significance was assessed using Spearman's correlation (SC), Fisher's exact test (a), and chi-square test (b), with a significance defined as a probability (p) value of ≤0.05. Under consideration of the Bonferroni correction, the p-value for significance is ≤0.008.

3.2.1.1. F229L mutation. The F229L variant was found in two male probands (probands #3 and #27). Proband #3 had early onset GTCSs. The parents of the 62-year-old proband #3 were reported to be healthy; however, their DNA was not accessible. Deoxyribonucleic acid of relatives of individual #27 who reported an affected sister with IGE with GTCSs on awakening was not available. Patients without F229L less frequently had early onset GTCSs (p = 0.001b), and, although not significant, a lower rate of IGE family history (OR: 4.6) as well as seizures that are provoked by sleep arousal (OR: 3.3) or alcohol consumption (OR: 5.5).

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Table 1 Detailed clinical data of all patients included in the study . Patient no.

Age

Sex

EO (y)

Sz types

Outcome (N6 m)

First sz

JME with ABSs

History of CAE/JAE

Seizureprovoking factors

GTCSs preceded by BMSs

SE

FH of JME/IGE

PPR

Early GTCSs (b12 years)

BMSs in series

Comments

1 2 3 4 5 6 7

73 77 62 72 42 45 55

F F M F F M F

6 12 11 16 8 2 23

2 2 1 1 3 2 3

SF+ SF+ SF− NSF+ NSF+ NSF+ SF+

ABSs BMSs GTCSs GTCSs ABSs ABSs ABSs

+ + − − + + +

+ − − − + + −

SD, ST SD SD, AL, ST − SD, ST − −

− + − − No − No

+ (ABSs) − − − − − −

JME/IGE − − IGE JME JME JME

− + − − − + −

− − + − No − No

− − − + − − −

8 9 10 11 12 13 14 15 16 17 18 19 20 21

50 78 43 48 55 43 45 41 49 21 27 27 35 22

F M F F M M M M F F F F F F

14 13 11 16 6 9 15 15 17 18 8 6 15 15

1 2 1 1 2 2 1 1 2 2 2 2 1 2

SF+ SF+ SF− NSF+ NSF+ SF+ SF+ NSF+ NSF+ SF+ SF+ SF+ SF+ NSF+

BMSs ABSs GTCSs GTCSs ABSs BMSs BMSs BMSs GTCSs BMSs ABSs ABSs GTCSs ABSs

− + − − + + − − + + + + − +

− + − − + − − − − − + + − −

SD, ST SD, AL SD, AL SD SD SD, AL SD, ST ST ST − SD, ST SD, ST SD, ST SD, ST

− − + + + − − − + + − − − −

− − + (BMSs) − − − − − − − − − − −

− − JME/IGE JME − JME JME − − − − IGE − JME/IGE

+ + + + + + + + − − − + − −

− − + − − − − − − − − − − −

− − + + + + + − + + + + − −

22 23 24 25 26 27 28 29

40 25 41 66 28 45 21 28

F F F F F M F F

19 19 14 16 13 19 7 12

1 0 3 2 2 3 2 2

NSF+ SF+ NSF+ NSF+ NSF+ SF+ SF+ SF+

BMSs BMSs ABSs GTCSs GTCSs BMSs ABSs ABSs

− − + + + + + +

− − + − − − + −

− SD, AL − SD SD, ST − SD SD, AL, ST

+ No No No + No − +

− − − − − − − −

− − − − − IGE JME JME

+ − − + + − + u

− No No − − No − −

+ + − U + − − +

30 31 32 33 34 35 36 37 38 39 40 41

20 22 25 26 51 33 25 26 27 18 30 28

F F F M M F M F M M F F

10 12 18 16 15 27 14 15 19 13 13 12

2 2 2 2 1 2 0 1 1 2 2 2

NA NSF+ NSF+ NSF+ NA NSF+ SF+ SF+ NA NSF+ SF+ SF+

ABSs GTCSs GTCSs GTCSs GTCSs ABSs BMSs BMSs BMSs GTCSs GTCSs ABSs

+ + + + − + − − − + + +

+ − − − − − − − − − − +

SD, ST − SD, ST − SD, AL, ST − − SD, ST SD, ST SD, ST SD SD

U − + − + U No + + + − −

− − − − − − − − − − − −

JME − − − − − JME IGE − IGE − −

− − − + − − − − − − − +

− + − − − − No − − − − −

− − + − + − + − − + − +

− − − − − − Sister of index patient #13 − − − − − − − − − − − − − Daughter of index patient #14 − − − − − − − Half sister of index patient #30 − − − − − − − − − − − −

ABSs — absence seizures; AED — antiepileptic drug; AL — alcohol consumption; BMSs — bilateral myoclonic seizures; CAE/JAE — childhood absence epilepsy/juvenile absence epilepsy; EO — epilepsy onset; F — female; FH — family history; GTCSs — generalized tonic–clonic seizures; IGE — idiopathic generalized epilepsy; JME — juvenile myoclonic epilepsy; M — male; NA — nonassessable; NSF+/− = nonseizure-free with/without AED treatment; PPR — photoparoxysmal response; SD — sleep deprivation; SE — status epilepticus; SF+/− = seizure-free with/without AED treatment; ST — stress; sz — seizure; seizure types: 0 — BMSs, 1 — BMSs + GTCSs, 2 — BMSs + GTCSs + ABSs, 3 — BMSs + ABSs; + = yes; − = no; U — unknown.

3.2.1.2. R294H variant. The R294H variant was found in a male proband and in a female proband; both of them developed JME from CAE (probands #6 and #30) and one of them additionally had PPRs Table 2 Frequency of long-term seizure outcome predictors in patients with JME. Outcome predictor

Frequency (n / 41)

PPR CAE developing to JME Status epilepticus GTCSs preceded by BMSs BMSs in series FH of JME FH of IGE other than JME FH of both JME and other IGE syndromes

17 (41.5%) 11 (26.8%) 2 (4.9%) (ABSs/BMSs) 14 (34.1%) 19 (46.3%) 10 (24.4%) 5 (12.2%) 3 (7.3%)

BMSs — bilateral myoclonic seizures; CAE — childhood absence epilepsy; FH — family history; GTCSs — generalized tonic–clonic seizures; JME — juvenile myoclonic epilepsy; IGE — idiopathic generalized epilepsy; PPRs — photoconvulsive responses.

(proband #6). At first sight, R294H appeared to be associated with early epilepsy onset (≤ 10 years), a subtype of JME developing from CAE, and a positive family history of JME. However, only one (proband #30, II/2 in Fig. 1) of two affected half sisters and her unaffected father turned out to carry the R294H variant, while the affected mother (one GTCS after sleep deprivation in 1998, PPR positive in one EEG) and the other half sister (#29, II/1 in Fig. 1) carried wild-type alleles. Consequently, lack of segregation in this family excluded R294H from further consideration. A sister of individual #6 is reported to have JME; however, DNA was not available. 3.2.1.3. R182H variant. The R182H variant was found in two female probands and in one male proband. Proband #10 had PPRs, GTCSs preceded by BMSs, BMSs in series, and status epilepticus, whereas proband #1 developed JME from CAE and had status epilepticus at least once during the course of the epilepsy. Proband #33 had ABSs and PPRs. In cases of JME with GTCSs, R182H negativity was associated with late GTCS onset (N12 years) (OR: 7.5), whereas R182H was associated with an

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Fig. 1. Pedigree of a family affected by JME: only one (II/2, #30) of the two affected half sisters and the unaffected father (I/3) turned out to carry the R294 variant, while the affected mother (I/2; one generalized tonic–clonic seizure after sleep deprivation and photoparoxysmal responses in one EEG) and the other half sister (II/1, #29) carried wild-type alleles.

increased risk of status epilepticus (p = 0.004a), a family history of IGE (p = 0.032b; OR: 10.7) and JME (OR: 4.9), as well as an increased risk of PPR positivity (OR: 2.9). 4. Discussion The present study supports the observation that EFHC1 mutations are not a common cause of familial JME in European populations. Annesi et al. [32] identified three EFHC1 missense mutations in 27 Italian families, while we found two in 38 German probands. Interestingly, F229L was found twice in both cohorts. In addition, recurrent mutational events leading to F229L in two Mexican families of the 44 families analyzed by Suzuki et al. [20] appear to strengthen the notion that F229L might be slightly enriched in individuals affected with JME. Variable penetrance of F229L has been described [20,34]. Asymptomatic carriers of F229L had either 3.5- to 6-Hz polyspike–wave complexes or even normal EEG [20]. While homozygosity of F229L has been reported to be associated with intractable epilepsy of infancy in two sibs of one Moroccan-Jewish family, their heterozygous sibs and parents appeared to be asymptomatic [34]. With regard to seizure outcome predictors investigated in our study, F229L appears to be associated with milder subtypes of JME as PPRs, GTCSs preceded by BMSs, BMSs in series, and a history of CAE/JAE, which were previously shown to be predictive of a worse long-term

outcome, could not be observed among our F229L probands. In contrast, probands with either R182H or R294H had a history of at least one relevant predictor (Table 3) indicating more severe courses of JME. Present knowledge suggests GTCSs, ABSs, and PPRs to be essential for predicting the outcome in patients with JME. In the present study, several interesting aspects on associations of those predictors with EFHC1 variants have been found. The value of GTCSs as a predictor for the seizure outcome is reported inconsistently. Geithner et al. found no association between the patient's age at the first GTCSs and the clinical course, whereas Syvertsen et al. demonstrated a longer interval between BMS onset and GTCS onset to be associated with a trend to GTCS remission, suggesting that a later GTCS onset serves as a predictor for subsequent GTCS remission [16,18]. In our study, one of the three probands with early GTCS onset showed F229L. Taken together, our results implicate that – assuming JME with GTCSs – the evidence of F229L may be negative predictive for long-term GTCS remission, which itself is predictive of complete seizure remission [16]. In our cohort, both probands with R294H had a history of early onset ABSs (2 and 10 years of age, respectively). Martínez-Juárez et al. found a lower remission rate among patients with CAE evolving to JME compared with other IGE subsyndromes (including patients with JME with adolescent ABSs) [35]. These data confirm prior findings that JME developing from CAE can be considered as a subgroup of JME with a different outcome [15]. Assuming the existence of a CAE evolving to JME in both R294H probands, an association of R294H with this subtype of JME (p = 0.007) can be supposed. Our finding of associations of R294H with early epilepsy onset (≤10 years) in general (p = 0.018) should, however, be interpreted in consideration of the finding of an association with CAE in R294H positive patients. In contrast to the findings of Martínez-Juárez et al. that adolescent ABSs are not predictive of a lower remission rate [35], one recent study reported the manifestation of ABSs in general as being predictive of a worse long-term outcome [17]; however, no association with the investigated EFHC1 variants was found in our cohort. Two of our R182H positive probands showed PPRs (OR: 2.9), suggesting R182H was associated with an increased risk of PPR positivity, which is consistent with prior findings [32]. Although PPRs are reported to be not predictive of the long-term seizure outcome in JME [16,36], PPR positivity was shown to highly increase the risk of seizure relapse after AED discontinuation [16]. In a very recent study on PPRs, Koeleman et al. hypothesized a heterogeneous, presumably polygenic cause [37]; additionally, several prior studies revealed associations with candidate genes (NEDD4-22, TRPC4, and BRD2) [38–40]. Lorenz

Table 3 Clinical data of all patients with EFHC1 variants. Patient no.

EFHC1 variant

Age

Sex

Outcome (N6 m)

First sz

JME with ABSs

PPR

GTCSs preceded by BMSs

SE

Hist. of CAE/JAE

BMSs in series

Early GTCSs (b12 years)

Family history

1 10

R182H R182H

73 43

F F

SF+ SF−

ABSs GTCSs

+ −

− +

− +

+ (ABSs) + (BMSs)

+ −

− +

− +

33 6 30

R182H R294H R294H

26 45 20

M M F

NSF+ NSF+ NA

GTCSs ABSs ABSs

+ + +

+ + −

− − U

− − −

− + +

− − −

− − −

3

F229L

62

M

SF−

GTCSs













+

27

F229L

45

M

SF+

BMSs

+



No







No

Mother affected with IGE, DNA not available. Mother affected with IGE; brother 1 affected with IGE with ABSs/GTCSs; brother 2 affected with IGE with GTCSs; sister affected with JME − Sister with JME, denied genetic testing. Half sister with JME (#29): wt alleles; unaffected father: R294H heterozygote; affected mother: wt alleles. See Fig. 1. Parents reported to be unaffected, DNA not available. Sister with IGE with GTCSs on awakening, DNA not available.

ABSs — absence seizures; AL — alcohol consumption; BMSs — bilateral myoclonic seizures; CAE/JAE — childhood absence epilepsy/juvenile absence epilepsy; F — female; GTCSs — generalized tonic–clonic seizures; IGE — idiopathic generalized epilepsy; JME — juvenile myoclonic epilepsy; M — male; NA — nonassessable; NSF+/− = nonseizure-free with/without AED treatment; PPR — photoparoxysmal response; SD — sleep deprivation; SE — status epilepticus; SF+/− = seizure-free with/without AED treatment; ST — stress; sz — seizure; + = yes; − = no; U — unknown; wt — wild type.

F. von Podewils et al. / Epilepsy & Behavior 44 (2015) 61–66

et al. reported BRD2 gene variations to confer susceptibility to PPRs and, moreover, BRD2 to be considered as a susceptibility gene for JME since it is located at 6p21, which was found to be linked to both PPR and JME independently [38]. This leads to the conclusion that PPRs and JME share epileptogenic pathways [38]. Taken together, it is quite likely that, in addition to the previously described candidate genes (NEDD4-22, TRPC4, and BRD2), EFHC1 variants – notably R182H – may confer susceptibility to PPRs and, therefore, may be considered as an independent risk factor for seizure remission after AED discontinuation. Several limitations of our study need to be considered. Firstly, this study is a single center study with a relatively small group of 41 patients; therefore, the possibility of type 2 statistical errors that limit statistical validity should be considered, particularly with regard to further subgroup analyses. Notably, the number of patients with evidence of EFHC1 variants was very small, yielding high specificities and relatively low sensitivities. Therefore, the aim of this study can rather be to describe associations of EFHC1 variants with clinical factors as supporting parameters than to identify significant correlations. However, comparing our results with those of large future study groups of patients with JME would most likely strengthen the validity of our findings and should be one object of future studies [35]. Secondly, we only examined the EFHC1 gene in our probands with JME. Future panel and exome analyses may enable further dissection of the heterogenic causes of JME. Thirdly, further clinical features that were not part of our study protocol have been described previously and should be included in future analyses [41]. Lastly, the number of EFHC1 variations found among our patients is relatively small. Assuming polygenetic causes of JME [37], the validity of our results may be reduced. Despite the above limitations, our results corroborate the heterogeneity of JME as an electroclinical epilepsy syndrome and suggest that, in context with clinical factors, genetic factors may contribute to better predict the long-term seizure outcome in patients with JME. Additionally, our findings of associations between certain clinical long-term outcome predictors and EFHC1 variants, such as F229L, R182H, and/or R294H, may potentially increase the clinician’s ability and confidence to recommend different treatment options to patients with JME. The decision on both the individual AED treatment and the possibility and time of withdrawal from medication should depend on several previously identified predictors, which appear to be linked with EFHC1 as follows: (1) The evidence of either F229L, R182H, or R294H being associated with an increased risk of status epilepticus and early GTCS onset as well as a decreased risk of BMS in series. Concerning subgroup analyses our findings reveal that: (2) F229L positivity may be considered as being negatively predictive for long-term GTCS remission and complete seizure remission, respectively, as it was associated with an elevated risk of early GTCS onset. (3) The evidence of R182H being associated with an increased risk of PPRs which was shown to be associated with seizure recurrence after AED discontinuation. (4) R294H positivity increases the risk of early epilepsy onset (b 10 years of age) and is associated with a subtype of JME developing from CAE, which was previously reported to be predictive of a worse long-term outcome. Despite these findings, the majority of our patients showed no mutation in the EFHC1 gene, corroborating the genetic heterogenity of JME as a syndrome. However, our results suggest that missense mutations in EFHC1, while not the sole cause of JME, may have modifying effects on the phenotypic variability of the disease. Our findings may help generate hypotheses about associations of EFHC1 genotypes and the phenotypic variability of JME and, thus, may help plan analyses of phenotypically well-characterized families or future prospective studies with larger study groups using high-throughput sequencing in this challenging patient population. Ethical approval We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. This study has been approved by the appropriate

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ethics committee and has, therefore, been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. Funding This study received no industrial, governmental, or institutional funding or sponsorship. Acknowledgments We gratefully acknowledge the assistance of Professor Dr. med. G. Rabending and Dr. rer. nat. P. Kolyschkow, University of Greifswald, Germany. Competing interests None of the authors has any conflicts of interest to disclose. References [1] Janz D, Christian W. Impulsive-petit mal. Dtsch Z Nervenheilkd 1957;176:344–86. [2] Janz D. Die Epilepsien. Stuttgart, Germany: Georg Thieme Verlag; 1969. [3] Janz D. Epilepsy with impulsive petit mal (juvenile myoclonic epilepsy). Acta Neurol Scand 1985;72:449–59. [4] Janz D. Juvenile myoclonic epilepsy. Epilepsy with impulsive petit mal. Cleve Clin J Med 1989;56(Suppl.1):23–33. [5] Panayiotopoulos CP, Obeid T, Tahan AR. Juvenile myoclonic epilepsy: a 5-year prospective study. Epilepsia 1994;35:285–96. [6] Dreifuss FE. Juvenile myoclonic epilepsy: characteristics of a primary generalized epilepsy. Epilepsia 1989;30(Suppl. 4):1–7 [24–27]. [7] Asconape J, Penry JK. Some clinical and EEG aspects of benign juvenile myoclonic epilepsy. Epilepsia 1984;25:108–14. [8] Delgado-Escueta AV, Enrile-Bacsal F. Juvenile myoclonic epilepsy of Janz. Neurology 1984;34:285–94. [9] Commission on Classification and Terminology of the International League Against Epilepsy. Proposal for revised classification of epilepsies and epileptic syndromes. Epilepsia 1989;30:389–99. [10] Usui N, Kotagal P, Matsumoto R, Kellinghaus C, Lüders HO. Focal semiologic and electroencephalographic features in patients with juvenile myoclonic epilepsy. Epilepsia 2005;46:1668–76. [11] Wolf P, Goosses R. Relation of photosensitivity to epileptic syndromes. J Neurol Neurosurg Psychiatry 1986;49:1386–91. [12] Doose H, Waltz S. Photosensitivity — genetics and clinical significance. Neuropediatrics 1993;24:249–55. [13] Verrotti A, Tocco AM, Salladini C, Latini G, Chiarelli F. Human photosensitivity: from pathophysiology to treatment. Eur J Neurol 2005;12:828–41. [14] Lu Y, Waltz S, Stenzel K, Muhle H, Stephani U. Photosensitivity in epileptic syndromes of childhood and adolescence. Epileptic Disord 2008;10:136–43. [15] Camfield CS, Camfield PR. Juvenile myoclonic epilepsy 25 years after seizure onset: a population-based study. Neurology 2009;73:1041–5. [16] Geithner J, Schneider F, Wang ZI, Berneiser J, Herzer R, Kessler C, et al. Predictors for long-term seizure outcome in juvenile myoclonic epilepsy: 25–63 years of followup. Epilepsia 2012;53:1379–86. [17] Senf P, Schmitz B, Holtkamp M, Janz D. Prognosis of juvenile myoclonic epilepsy 45 years after onset: seizure outcome and predictors. Neurology 2013;81(24): 2128–33. [18] Syvertsen MR, Thuve S, Stordrange BS, Brodtkorb E. Clinical heterogeneity of juvenile myoclonic epilepsy: follow-up after an interval of more than 20 years. Seizure 2014;23(5):344–8. [19] Delgado-Escueta AV. Advances in genetics of juvenile myoclonic epilepsies. Epilepsy Curr 2007;7(3):61–7. [20] Suzuki T, Delgado-Escueta AV, Aguan K, Alonso ME, Shi J, Hara Y, et al. Mutations in EFHC1 cause juvenile myoclonic epilepsy. Nat Genet 2004;36:842–9. [21] Medina MT, Suzuki T, Alonso ME, Durón RM, Martínez-Juárez IE, Bailey JN, et al. Novel mutations in myoclonin1/EFHC1 in sporadic and familial juvenile myoclonic epilepsy. Neurology 2008;70:2137–44. [22] Stogmann E, Lichtner P, Baumgartner C, Bonelli S, Assem-Hilger E, Leutmezer F, et al. Idiopathic generalized epilepsy phenotypes associated with different EFHC1 mutations. Neurology 2006;67:2029–31. [23] Suzuki T, Miyamoto H, Nakahari T, Inoue I, Suemoto T, Jiang B, et al. Efhc1 deficiency causes spontaneous myoclonus and increased seizure susceptibility. Hum Mol Genet 2009;18(6):1099–109. [24] De Nijs L, Wolkoff N, Coumans B, Delgado-Escueta AV, Grisar T, Lakaye B. Mutations of EFHC1, linked to juvenile myoclonic epilepsy, disrupt radial and tangential migrations during brain development. Hum Mol Genet 2012;21(23):5106–17. [25] De Nijs L, Wolkoff N, Grisar T, Lakaye B. Juvenile myoclonic epilepsy as a possible neurodevelopmental disease: role of EFHC1 or Myoclonin1. Epilepsy Behav 2013; 28(Suppl.1):58–60. [26] Waltz S, Christen HJ, Doose H. The different patterns of photoparoxysmal response — a genetic study. Electroencephalogr Clin Neurophysiol 1992;83:138–45.

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Predictive value of EFHC1 variants for the long-term seizure outcome in juvenile myoclonic epilepsy.

This study aimed to determine the contribution of EFHC1 variants to the phenotypic variability of juvenile myoclonic epilepsy (JME) and to evaluate th...
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