Early clinical features in Dravet syndrome patients with and without SCN1A mutations

Epilepsy Research (2012) 99, 21—27

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Early clinical features in Dravet syndrome patients with and without SCN1A mutations Cristina Petrelli a,∗, Claudia Passamonti b, Elisabetta Cesaroni b, Davide Mei c, Renzo Guerrini c, Nelia Zamponi b, Leandro Provinciali a a

Neurology Clinic, Polytechnic University of Marche, Ancona, Italy Child Neurology Unit Department, Ospedali Riuniti, Ancona, Italy c Pediatric Neurology Unit and Laboratories, Children’s Hospital A. Meyer — University of Florence,Italy b

Received 15 December 2010; received in revised form 3 October 2011; accepted 9 October 2011 Available online 8 November 2011

KEYWORDS Dravet syndrome; SCN1A mutations; Seizures

Summary Background: SCN1A is the most clinically relevant epilepsy gene, most mutations causing Dravet syndrome (also known as severe myoclonic epilepsy of infancy or SMEI). We evaluated clinical differences, if any, between young patients with and without a SCN1A mutations and a definite clinical diagnosis of Dravet syndrome. Methods: Twenty-five patients with a diagnosis of Dravet Syndrome (7 males, 18 females; mean age at inclusion: 10.3; median: 9 ± 7; range: 18 months—30 years) were retrospectively studied. A clinical and genetic study focusing on SCN1A was performed, using DHPLC, gene sequencing and MLPA to detect genomic deletions/duplications. A formal cognitive and behavioral assessment was available for all patients. Results: Analysis revealed SCN1A mutations comprising missense, truncating mutations and genomic deletions/duplications in eighteen patients and no mutation in seven. The phenotype of mutation positive patients was characterized by a higher number of seizures/month in the first year of life, an earlier seizure onset and a higher frequency of episodes of status epilepticus. The cognitive and behavioral profile was slightly worst in mutation positive patients. Conclusions: These findings confirm that SCN1A gene mutations are strongly associated to a more severe phenotype in patients with Dravet syndrome. © 2011 Elsevier B.V. All rights reserved.

Introduction

∗ Corresponding author at: Neurology Clinic, Polytechnic University of Marche Ospedali Riuniti, Via Conca, Ancona, Italy. Tel.: +39 0715962484; fax: +39 0715962502. E-mail address: [email protected] (C. Petrelli).

Dravet syndrome is a rare and distinct epileptic encephalopathy (Commission on Classification and terminology of the International League Against Epilepsy, 1989), which begins with infantile onset of febrile hemiclonic status epilepticus and evolves to a pattern of multiple seizure types including focal, myoclonic, absence,

0920-1211/$ — see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.eplepsyres.2011.10.010

22 and atonic seizures, associated with marked slowing or stagnation of psychomotor development, often accompanied by behavioral disturbances (Wolf et al., 2006; Riva et al., 2009; Ragona et al., 2010; Chieffo et al., 2010). Neuroimaging studies and metabolic investigations are not contributory, suggesting no structural or interposed metabolic abnormality. The SCN1A gene, which encodes the ␣1 subunit of the neuronal sodium channel, is the most relevant epilepsy gene with the largest number of epilepsy-related mutations (Escayg et al., 2001; Wallace et al., 2001; Marini et al., 2007; Harkin et al., 2007) including genetic (generalized) epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome. In the mouse genetic model of Dravet syndrome a selective failure of excitability of ␥-aminobutyric acid (GABA)ergic neurons, resulting from SCN1A mutations, has been shown (Yu et al., 2006). The SCN1A gene has also been involved in other phenotypes such as familial hemiplegic migraine and other non-epileptic disorders (Gambardella and Marini, 2009). The SCN1A frequency of missense and truncating mutations is approximately equal, both accounting for 40% of all mutation-positive patients (Scheffer, 2011). Interestingly, the large majority of the SCN1A mutations causing Dravet syndrome results in loss of function, whereas the GEFS+ phenotype is usually observed in patients harboring missense substitutions. SCN1A mutations can be identified in about 70—80% of patients with Dravet syndrome (Claes et al., 2001, 2003; Sun et al., 2008; Harkin et al., 2007; Mulley et al., 2005; Kanai et al., 2004; Fujiwara et al., 2003; Sugawara et al., 2002; Ohmori et al., 2002). Furthermore, recent studies have demonstrated that about 12% of mutation-negative Dravet syndrome patients have SCN1A genomic rearrangements detectable by multiplex ligation-dependent probe amplification (MLPA) and/or by Array-CGH (Marini et al., 2010). Some authors (Depienne et al., 2009; Marini et al., 2010) identified alterations in the PCDH19 gene (encoding protocadherin 19) in patients who were negative for mutation or rearrangements of the SCN1A gene. Those patients had very similar clinical features to SCN1A-positive patients, indicating that the two clinical spectra largely overlap. Remarkable phenotypic variability exists between patients with SCN1A mutations, even within the same family (Suls et al., 2010; Guerrini et al., 2010). The occurrence of possible between SCN1A mutated and non mutated patients, as well as between Dravet patients with SCN1A mutations and Dravet patients with PCDH19 alterations, has been the object of recent investigations (Depienne et al., 2009; Marini et al., 2007, 2010). A study of Marini et al. (2007) revealed that Dravet patients with SCN1A mutations had an earlier age of onset of febrile seizures. We evaluated whether clinical differences exists between Dravet syndrome patients with SCN1A alterations and those without. To this purpose, medical reports from twenty-five patients with Dravet syndrome have been retrospectively reviewed.

C. Petrelli et al. clinical details and DNA were available, were reviewed from the Child Neurology Department of ‘‘G. Salesi Hospital’’. The research received prior approval by the local ethical committee and each parent/guardian signed an informed consent form. Mean age at the time of the study was 10.3 years (median: 9 ± 7, range: 18 months—30 years). All patients fulfilled the following diagnostic criteria established by the International League Against Epilepsy (Commission on Classification and terminology of the International League Against Epilepsy, 1989): normal development before seizure onset; occurrence of either generalized, unilateral, or partial seizures during the first year of life; seizures that were frequently provoked by fever; presence of myoclonic seizures with spike and wave-complex or segmental myoclonus, diffuse spike-waves or focal spikes on EEG during the clinical course; intractable epilepsy; gradual evidence of psychomotor delay after two years of age. For each patient the following clinical features were retrospectively studied: mean age at onset of first febrile or afebrile seizure, family history in first degree relatives, number of seizures before one year of age, seizure types (generalized tonic-clonic seizures, hemiconvulsion and focal seizure, status epileptic), epileptic discharges on EEG. Patients’ demographical and clinical details are summarized in Table 1. Seizure type and number were established by reviewing seizure diaries given to patients and medical reports.

Genetic analysis Molecular analysis was performed on genomic DNA extracted from blood using standard procedures. All 26 exons of SCN1A were amplified by polymerase chain reaction (PCR) and analyzed by DHPLC as previously described (Marini et al., 2007). Exons showing abnormal DHPLC chromatographic profiles were analyzed by direct sequencing. Patients negative to DHPLC screening were first sequenced to ensure that point mutations were not missed, and then screened by MLPA. When an alteration was found, genetic test of parents was requested in order to search for inherited mutations. The novel SCN1A missense substitutions were not found in a cohort of 95 control DNA (195 alleles). SCN1A-negative patients were screened by MLPA, using the P137-A2 SCN1A kit of MRC Holland (Wang et al., 2008; Marini et al., 2009), in order to detect genomic deletions/duplications. In SCN1Anegative female patients the six exons covering the coding regions of PCDH19 and their intron—exon boundaries were PCR amplified and sequenced as previously described (Marini et al., 2010). Patients were divided in two groups on the basis of presence/absence of SCN1A mutations: the case group presented SCN1A-mutation (SCN1A+), while the control group had no SCN1A-mutation (SCN1A−) (Table 2).

Cognitive and behavioral evaluation

Methods Subjects Medical reports of twenty-five patients with Dravet syndrome (eighteen females and seven males), for whom

A formal cognitive and behavioral examination, including developmental quotient (DQ) and adaptive behavior was available for all patients. Assessment was performed at two sessions, i.e. at the time of diagnosis (T1), which was performed between 18 and 30 months of life (data available

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 P21 P22 P23 P24 P25

Patients’clinical and demographic details. Sex

Age at onset (months)/Age at inclusion (years)

Result of genetic test

Epileptic discharges on EEG

No. of seizures before one year of age

Status epilepticus

Family history in first-degree relatives

Seizure types

AED*

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

11/10 12/6 5/12 4/6 8/30 8/10 5/6 1/6 6/9 7/6 7/12 5/6 4/14 4/14 4/8 2/18 months 8/9 4/10 13/6 7/8 4/28 8/11 3/16 5/6 6/6

+ − + + + − − − + + + + + + + + + + − − + + + − +

+ + + − + + + − + + − + + + + + + + + − + + + + +

1 0 7 9 32 6 1 1 4 13 60 13 N/A 12 40 100 80 6 0 4 80 1 1 N/A 15

+ − + + + + + − + + + + − + + + − − − − + + + − +

− + + − − − − − − + + + − − − − − − + + + − + − +

FS, H GTC FS, H FS, H GTC FS, GTC FS GTC FS, H FS FS GC FS GTC, H GTC, H GTC, FS GTC GTC GTC FS, GTC FS GTC, FS GTC GTC, FS, H GTC, FS, H

DPA, TPM DPA, LEV, NZP DPA, TPM, CLB DPA, TPM LEV, ZNS, AZM DPA DPA, TPM DPA LEV, DPA, TPM DPA, DINT, NZP AZM, DPA, TPM OXC, LEV, RIV DPA, TPM, LEV DPA, NZP LEV, DPA, ZNS, LZP DPA, CLB TPM, AZM, NZP + KD DPA, LEV, TPM TPM DPA PB, ETS, LTG, NZP DPA, TPM, CNZ ZNS, DPA, CLB DPA, AZM TPM, LEV, CLB

Early clinical features in Dravet syndrome patients with and without SCN1A mutations

Table 1

GTC: Generalized tonic-clonic seizure; GC: generalized clonic seizure; FS: focal seizures; H: hemiconvulsions; +: SCN1A mutation positive; −: SCN1A mutation negative; * Antiepileptic drugs: AZM: acetazolamide; CBZ: carbamazepine; CLB: clobazam; CNZ: clonazepam; VPA: valproic acid; PHT: phenytoin; ETS: ethosuximide; FLB: felbamate; GBP: gabapentin; LEV: levetiracetam; LTG: lamotrigine; LZP: lorazepam; NZP: nitrazepam; KD: ketogenic diet; OXC: oxcarbazepine; PB: phenobarbital; PRM: primidone; STM: sulthiame; TGB: tiagabine; TPM: topiramate; VGB: vigabatrin.

23

24 Table 2

C. Petrelli et al. Patients’ genetic mutations. SCN1A DHPLC and sequencing analysis

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 P21 P22 P23 P24 P25

cDNA

Protein

c.893 894del Negative Negative c.5435G > A c.602 + 1G > A Negative Negative Negative c.5260G > A c.1170 + 1 G > T c.5641G > A c.5347G > A c.3852delG c.697 698del c.1154A > G c.5348C > T N/A c.5536 5539del Negative Negative c.302G > A c.4073G > C Negative Negative c.2928G > A

p.Val298GlufsX3

p.Trp1812X p.?

p.Gly1754Arg p.? p.Glu1881Lys p.Ala1783Thr p .Trp1284CysfsX7 p.Leu233GlufsX43 p.Glu385Gly p.Ala1783Val N/A p.Lys1846SerfsX11

p.Arg101Gln p.Trp1358Ser

p.Met976Ile

SCN1A MLPA

Transmission

NT Negative c.265-? c.602+?del NT NT Negative Negative Negative NT NT NT NT NT NT NT NT N/A NT Negative Negative NT NT c.1377+? c.2416-?dup N/A NT

Parents not available

Novel

Mother De novo De novo

Guerrini et al. (2010) Novel Fujiwara et al. (2003)

De novo De novo Parents not available Mother (mosaicism) De novo De novo De novo De novo

Novel Novel Novel Harkin et al. (2007) Novel Novel Novel Marini et al. (2007)

De novo

Claes et al. (2001)

De novo Parents not available Parents not available

Fukuma et al. (2004) Zucca et al. (2008) Novel

Parents not available

Novel

for all patients), and at the admission to elementary school (T2), which was at six years (data available for all patients except P16). Five patients (all from case group) were followed-up through the teenage years. Cognitive functioning was evaluated using the Italian version of Stanford—Binet Scale (Bozzo and Mansueto Zecca, 1968) for patients from 2 to 6 years old, and the Weschler scales (WISC-R) (Wechsler, 1949) for patients from 6 to 16 years. Cognitive delay was classified as ‘‘severe’’, ‘‘moderate’’ or ‘‘mild’’, when the DQ was below 40, between 40 and 55, and between 55 and 70, respectively. Adaptive behavior, including four domains (communication, daily living skills, socialization, and motor skills) was measured using the Italian version of Vineland Adaptive Behavior Scales (VABS) (Balboni and Pedrabissi, 2003). This instrument is a semi-structured interview addressed to parents, which provides normative scores (mean: 100; standard deviation: 15) for children from 0 to 18 years of age, with higher scores indicating better functioning.

Data analysis Differences in the frequency of distribution of clinical and electroencephalographic features between SCN1A+ and SCN1A− were evaluated by the chi-square test. An independent sample T-test was used to compare differences in the mean age of seizure onset between the two groups.

Mann—Whitney U test was performed to compare cognitive and behavioral scores between SCN1A+ and SCN1A−. A pvalue < 0.05 was considered statistically significant.

Results Genetic analysis DHPLC and sequencing analysis detected sixteen SCN1Amutations, including twelve missense mutation and four truncating mutations (two frame-shift and two non-sense mutations). MLPA identified two patients (P3, P23; see Table 1) harboring SCN1A genomic rearrangements. A deletion c.265-? c.602+?del encompassing three SCN1A exons (2—4) was found in the first patient, while a duplication c.1377+? c.2416-?dup was found in the second one. The patient 3’s deletion was also found in her brother, mother, uncle and grandfather. Only patient 3 and her uncle exhibited Dravet syndrome. This family has been previously published (Guerrini et al., 2010). No female patients with PCDH19 mutations were found (see Table 2). Thus, genetic analysis identified eighteen patients with SCN1A mutations, i.e. group SCN1A+ (72%), and seven patients without mutation, i.e. group SCN1A− (28%). Compared to SCN1A+, group SCN1A− was characterized by a significantly higher number of seizures before one year of age (case: 27.8 ± 32.5; control: 2 ± 2.4, p = 0.04), an higher frequency of patients with

Early clinical features in Dravet syndrome patients with and without SCN1A mutations

25

Table 3 Clinical details in SCN1A− and SCN1A+. The number and frequency of subjects SCN1A negative (SCN1A−) and SCN1A positive (SCN1A+) is reported in columns 2 and 3. The p value for each statistical comparison is reported in column 4. Asterisks denote significance. Patients’ characteristics at onset

SCN1A negative (n:8)

SCN1A positive (n:17)

p-value (˛ = 0.05)

Mean age at onset (months) of first seizure Family history in first-degree relatives Number of seizures before one year of age (mean) GTC,GC Focal seizure and hemiconvulsions Status epilepticus Epileptic discharges on EEG

7.3 ± 4.2 3 (43%) 2 ± 2.4 6 (85%) 4 (57%) 2 (28%) 5 (71%)

5.5 ± 2.2 7 (39%) 27.8 ± 32.5 10 (55%) 13 (72%) 15 (83%) 16 (88%)

0.19 0.8 0.04* 0.03* 0.6 0.008* 0.30

first seizure onset before 7 months of life (case group: 77%; control group = 57%, p = 0.03), and a higher frequency of status epilepticus (case: 83%; control: 28%, p = 0.008). Patients of case group presented an earlier mean age of seizure onset (5.5 ± 2.2) than those of the control group (7.4 ± 4.2, p = 0.19) and a higher frequency of focal seizures and hemiconvulsions (72% vs. 57%, p = 6) although differences were not significant. As for seizure types, we found that 55% of SCN1A+ patients presented tonic-clonic or clonic seizures vs. 85% of SCN1A− patients (p = 0.003). Epileptic discharges on EEG did not show a significant difference between groups (SCN1A+: 88%; SCN1A−: 71%; p = 0.3). Results of the statistical analysis are summarized in Table 3.

Cognitive and behavioral outcome Before epilepsy onset, psychomotor development and behavior were reported as being normal in all patients. At time of diagnosis (range of age: 18—30 months) SCN1A+ patients showed a lower DQ (mean score: 57; st.dev: 22), ranging from severe (n:2) and moderate mental retardation (n:8) to borderline (n:6) and normal level (n:2), compared to SCN1A− patients (mean score: 74; st.dev: 15), who ranged from mild mental retardation (n:2) to borderline (n:3) and normal level (n:2). This difference was close to significance (p = 0.05) (see Table 2). All patients showed an impairment in the adaptive behavior, especially in the area of communication and socialization, with a lower mean score in case group (mean VABS score: 56, st.dev: 24) compared to controls (mean VABS score: 68, st.dev: 13, p = 0.04). At admission to the primary school, a slight decline in cognitive level was observed in all patients, and the difference between the two groups became more apparent; SCN1A+ patients (mean DQ = 49, st.dev: 21; mean VABS score: 50, st.dev: 21) experienced a greater decline in cognitive profile and adaptive behavior than SCN1A− patients (mean DQ = 69, st.dev: 14; mean VABS score: 65, p = 0.03) (see Table 2). Compared to T2, the five patients who were assessed during the teenage years (T3) (range of age: 12—15 years) showed a stagnation of their cognitive level. However, four out of the latter patients reported a significant improvement in their adaptive behavior, especially in the area of socialization and daily life skills, with an increase between five and eight points in VABS standard score.

Discussion In this study we tried to identify the clinical features associated to SCN1A mutation in Dravet syndrome. Seventy-two percent of patients with Dravet syndrome had a SCN1A alteration demonstrated after DHPLC/sequencing and MLPA. This frequency distribution is in line with previous studies, which found SCN1A mutations in 70—80% of Dravet syndrome patients (Claes et al., 2001, 2003; Sun et al., 2008; Harkin et al., 2007; Mulley et al., 2005; Kanai et al., 2004; Fujiwara et al., 2003; Sugawara et al., 2002; Ohmori et al., 2002) and SCN1A genomic rearrangements in 12% of mutation-negative Dravet syndrome patients (Marini et al., 2010). Other clinical features did not result significantly different between SCN1A+ and SCN1A−, for example the mean age of onset of first febrile or afebrile seizure. However, an early onset of seizures was observed in SCN1A+ compared to SCN1A−, according to other studies which reported this clinical feature in a wide population of SCN1A+ patients including both GEFS+ and Dravet syndrome phenotype (Kanai et al., 2004; Sijben et al., 2009). Moreover, no significant difference was observed regarding family history in first degree-relatives between the two groups. We identified three cases in which the mutation of SCN1A gene was found in one parent. One female patient (P12, see Table 1) had the same missense mutation of her mother (c.5347G > A, Ala1783Thr), but their phenotypes were different (Dravet syndrome in the daughter and GEFS+ in the mother). In another patient (P25; see Table 1), the same mutation (c.2928G > A; p.Met976Ile) was found in the father and in the twin-sister; the father had febrile and afebrile seizure during his infantile age, which disappeared at 12 years old, while her twin-sister had only one febrile seizure. MLPA identified one patient (P3; see Table 1) with a deletion of three exons (2—4) of the SCN1A gene. Patient 3’s deletion was also found in her brother, mother, uncle and grandfather. Only patient 3 and her uncle exhibited Dravet syndrome (Guerrini et al., 2010). These results are in line with a study from Depienne et al. (2010), which demonstrated that patients harboring SCN1A de novo mutations may either have parents carrying the mutation in a mosaic fashion (about 7% of families with Dravet syndrome) or be part of multiplex GEFS+ families. Finally, our results indicate lack of association between SCN1A mutations and increased incidence and age at onset of EEG discharges.

26 Neuropsychological assessment identified a variable degree of cognitive decline in both SCN1A+ and SCN1A− patients between 18 months of life and six years of age, while a stagnation was observed during the teenage years in mutation positive patients. More interestingly, a greater delay in cognitive development and adaptive behavior was observed in SCN1A+ patients compared to controls, which was particularly evident at admission to primary school. Several factors can account for these results, such as a higher seizure frequency before one year of life and an early seizure onset in mutation positive patients. Moreover, it should be evoked a role of other factors other than epilepsy activity. It is worth noting, in fact, that the genetic mutation (SCN1A) with a loss of function in Nav1.1 (a voltage-gated sodium channel lacking in Dravet syndrome) may play a role in determining a general developmental delay (Schmahmann, 2004). As for the long-term cognitive evolution of mutation positive patients, it seems crucial to consider both the effects of antiepileptic treatments for seizure control (Zamponi et al., 2010), and that the impact of rehabilitation programs, which are early performed in patients with a developmental delay. Both these factors might have preserved further cognitive deterioration and should be taken into account in future longitudinal research. Because of the small sample size and the numerical difference between SCN1A+ and SCN1A− patients, no confident conclusion can be drawn about a possible different cognitive outcome between mutation positive and negative patients. However, the present observations reveal a possible worst cognitive and behavioral development in Dravet syndrome patients with SCN1A mutation, in line with previous reports from literature (Wolf et al., 2006; Riva et al., 2009; Ragona et al., 2010) and suggest the importance of performing an early cognitive and neuropsychological assessment to orient clinical practice and contrast developmental delays (Chieffo et al., 2010). In conclusion, the present findings confirm that SCN1A gene mutations are strongly associated to a more severe phenotype in patients with Dravet syndrome. This phenotype is characterized by a higher number of seizures/month in the first year of life, an earlier seizure onset and a higher frequency of episodes of status epilepticus. In order to evaluate the prognosis of this phenotype in patients carrying a mutation, a long term follow-up is needed, in the context of an extensive polycentric survey. Despite the limitations of the present study, the quoted results confirm the high sensitivity of genetic analysis in the clinical practice of Dravet syndrome patients and highlights the complex relationships between genotype and phenotype in patients harboring SCN1A mutations.

Disclosure None of the authors has any conflict of interest to disclose. 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.

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