Referat zum Thema Diagnostische Studien Artikel: Hattori et al. (2008): A screening test for the prediction of before one year of age. Epilepsia, 49(4):626-633

Betreuer: Prof. Dr. Ulrich Stephani; Klinik für Neuropädiatrie: [email protected] Dipl. Inform. J. Hedderich; Institut für Medizinische Informatik und Statistik: [email protected]

Stichpunkte und Hinweise zur Gliederung des Referates: Krankheit: Fieberkrämpfe / Dravet-Syndrom – Definition, Ätiologie, Prävalenz, Behandlung, Krankheitsverlauf und Folgen. Diagnostik: Diagnostisches Vorgehen (Goldstandard), EEG, Genetik (PCR), prädiktive klinische Faktoren. Studienansatz: Motivation für die Studie, Studientyp, Ein- und Ausschlusskriterien bei der Patientenrekrutierung (Tabelle 1). Statistische Verfahren: Welche statistischen Methoden wurden zu welchen Fragen angewandt? Schwerpunkt exakter Fisher Test, ROC und Gütekriterien! Ergebnisse: a) Alter bei 1. Auftreten und Anzahl der Anfälle (Abbildung1). b) Gegenüberstellung der Faktoren (Tabelle 2). c) Diagnostische Bewertung (Tabelle 4). Diskussion: Die Modelle 1 – 4 (Abbildung 2); Kritische Bewertung der Studie hinsichtlich der verwendeten Methoden und statistischen Verfahren. Anwendbarkeit und Limitierung der Ergebnisse.

¾ Wichtig: Die aufgeführten Stichpunkte dienen nur zur Orientierung. Das Setzen von Schwerpunkten, der Aufbau des Referates und das eventuelle Einbringen von zusätzlichen Aspekten ist den Referenten überlassen. In allen Punkten muss dabei aber ein Bezug zur vorliegenden Studie hergestellt werden. Die Referenten werden gebeten, sich nach Lesen und Verstehen des Artikels (spätestens 2 Wochen vor dem Termin des eigenen Referates) bei den Dozenten zu melden, damit offene Fragen geklärt bzw. ergänzende Informationen weitergegeben werden können.

Epilepsia, 49(4):626–633, 2008 doi: 10.1111/j.1528-1167.2007.01475.x FULL-LENGTH ORIGINAL REASARCH

A screening test for the prediction of Dravet syndrome before one year of age ∗Junri Hattori, †Mamoru Ouchida, ∗Junko Ono, ‡Susumu Miyake, §Satoshi Maniwa, ¶Nobuyoshi Mimaki, ∗Yoko Ohtsuka, and ∗∗Iori Ohmori

Departments of ∗Child Neurology and †Molecular Genetics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan; ‡Kagawa Prefectural Central Hospital, Pediatrics, Takamatsu, Kagawa, Japan; §Matsuyama Red Cross Hospital, Pediatrics, Ehime, Japan; ¶Kurashiki Medical Center, Pediatrics, Okayama, Japan; and ∗∗Department of Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan

SUMMARY garded as significant risk factors for Dravet syn- Purpose: Our aim was to develop a screening test drome. Other factors highly predictive of this syn- to predict Dravet syndrome before the first birth- drome were hemiconvulsions, partial , my- day based on the clinical characteristics of infants oclonic seizures, and hot water–induced seizures. and the SCN1A mutation analysis. A total clinical score of six or above was the cut- Methods: Ninety-six patients who experienced off value indicating a high risk of Dravet syndrome. febrile seizures before the age of one were en- SCN1A missense and truncated mutations were de- rolled. The patients were divided into two groups— tected significantly more often in the Dravet syn- the Dravet syndrome group (n = 46) and the non- drome group than in the non-Dravet syndrome Dravet syndrome group (n = 50).Wecompared group. the clinical characteristics before one year of age Discussion: This simple screening test was designed of the two groups. We analyzed all coding exons of to be used by general pediatricians. It could help to the SCN1A gene by the direct sequencing method. predict Dravet syndrome before one year of age. Scores from 0 to 3 were assigned to each risk factor If the sum of the clinical risk score is ≥ 6, then based on the odds ratio and p-value. the performance of an SCN1A mutation analysis is Results: An age of onset of febrile ≤ 7 recommended. months, a total number of seizures ≥ 5, and pro- KEY WORDS: SCN1A, Dravet syndrome, SMEI, longed seizures lasting more than 10 min. were re- Screening test.

Febrile seizures (FS) are the most common type of characterized by the occurrence of FS before one year of seizure in childhood between five months to six years of age (Dravet, 1978). Dravet syndrome is diagnosed at two age. Their incidence rate ranges from 2% to 14% of all chil- to four years of age after the appearance of all of its clin- dren (Stanhope et al., 1972; Van den Berg, 1974; Hauser & ical features. Due to the recurring presence of prolonged Kurland, 1975; Nelson & Ellenberg, 1978; Tsuboi, 1984). febrile and afebrile seizures in early childhood, it is desir- FS spontaneously stop before six years of age; therefore, able that certified pediatric neurologists or it is generally accepted that treatment with an antiepilep- manage the patients with Dravet syndrome as early as pos- tic drug regimen is not required. Dravet syndrome (pre- sible. However, it is difficult for general pediatricians to viously termed SMEI) is an intractable form of distinguish Dravet syndrome from benign FS or epilepsy before one year of age because of their similar clinical fea- Accepted July 11, 2007; Online Early publication December 12, 2007. tures at onset. Address correspondence to Iori Ohmori, Department of Cellular Phys- In recent years, SCN1A mutations have been identified iology, Graduate School of Medicine, Dentistry and Pharmaceutical Sci- ences, Okayama University, 5–1 Shikata-cho, 2-chome, Okayama 700– in 30–80% of patients with Dravet syndrome (Claes et al., 8558, Japan. E-mail: [email protected] 2001; Ohmori et. al., 2002; Sugawara et al., 2002; Nab- Blackwell Publishing, Inc. bout et al., 2003; Wallace et. al., 2003; Fukuma et al., C 2008 International League Against Epilepsy 2004). The SCN1A mutations were also observed in 3.1

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Screening Test for Dravet Syndrome to 5.7% of patients with with FS be more frequently collected at such centers while benign plus (GEFS+) (Escayg et al., 2001; Wallace et al., 2001). cases with isolated FS tended to be excluded in this study. GEFS+ is a familial epilepsy syndrome characterized by heterogeneous phenotypes including FS, febrile seizures Methods plus (FS+), mild generalized , and severe epilep- The epileptic syndromes of the 96 patients at study en- tic encephalopathies (Scheffer & Berkovic, 1997). One rollment were divided into two groups-–the Dravet syn- SCN1A missense mutation has been reported in a type of drome group and the non-Dravet syndrome group. The familial (Mantegazza et al., 2005). It is not Dravet syndrome group includes typical Dravet syndrome clear whether a genetic test of the SCN1A gene is useful for and borderline Dravet syndrome patients. The patients with predicting Dravet syndrome at an early age. Dravet syndrome fulfilled the following criteria: normal We retrospectively investigated the risk factors in pa- development before seizure onset, the occurrence of ei- tients who experienced a febrile seizure before one year ther generalized, unilateral, or partial seizures during the of age. Clinical features and SCN1A mutations were an- first year of life, seizures that were frequently provoked alyzed to predict Dravet syndrome. The development of by fever, the presence of myoclonic seizures with spike- a risk score screening test may enable general pediatri- and-wave complex or segmental myoclonus, diffuse spike- cians to identify patients with Dravet syndrome at an early waves or focal spikes on EEG during the clinical course, stage. intractable epilepsy, and gradual evidence of psychomo- tor delay after two years of age. Epileptic conditions of the non-Dravet syndrome group were subclassified accord- ing to the diagnostic criteria of the International League SUBJECTS AND METHODS Against Epilepsy classification (2001) (ILAE, 2001). If the Participants patients had recurrent seizures or neurological signs, CT Individuals who experienced FS before one year of age or MRI brain scans were performed to exclude a focal were recruited from outpatient pediatric clinic of seven organic lesion. The mental developmental levels at study participating medical centers (Okayama university hospi- enrollment (the mean age was 11.6 years in non-Dravet tal, Tottori university hospital, Kagawa prefectural central syndrome and 17.1 years in Dravet syndrome) were clas- hospital, Kurashiki medical center, Hiroshima city hospi- sified into three groups—normal development, mild retar- tal, NKK Fukuyama hospital, and Matsuyama Red Cross dation, and moderate to severe retardation. We carried out hospital) during 2003 and 2004 for whom clinical details either the WISC-R, WISC-III, or Kaufman assessment bat- and DNA were obtainable. A case was determined to have tery for children for an assessment of psychomotor devel- a febrile seizure when a temperature equivalent to ≥38.5◦C opment for participants who had an imperfect ability in was measured within 1 h of the event. This definition ex- school performance. Participants who had a normal school cludes seizures that accompany neurologic illnesses, such performance or participants whose IQ were more than 70 as meningitis, encephalitis, or toxic encephalopathy. The were classified into the normal group. Participants whose criteria for inclusion in the study were the onset of FS be- IQ ranged 50 ≤ IQ < 70 were classified into the mild re- fore the first year of life, normal development before the tardation group. Participants whose IQ less than 50 were onset of seizures, and a follow-up period longer than three classified into the moderate-to-severe retardation group. years. The exclusion criteria included underlying neurolog- To determine the clinical predictive factors for Dravet ical disorders. One hundred and twelve participants were syndrome, we retrospectively studied the clinical data of assessed for eligibility by at least two child neurologists, participants before one year of age. The cases were system- and sixteen participants were thus excluded. atically evaluated based on an exhaustive review of medical The study protocol and informed consent documents records from certified child neurologists. We compared the were approved by the Ethics Committee of Okayama Uni- following clinical characteristics of the two groups: age at versity Graduate School of Medicine, Dentistry, and Phar- onset, sex, family history of a febrile seizure or epilepsy maceutical Sciences. Participants or parents provided writ- in first- or second-degree relatives, the total number of any ten informed consent before enrollment. type of seizures before one year of age, the absence or pres- The participants were recruited retrospectively. One of ence of a prolonged seizure longer than 10 min, seizure the inclusion criteria was a follow-up period longer than types, precipitating factors, EEG findings, and SCN1A mu- three years to know the outcome. At study enrollment, all tations. participants had already been followed at various medical An EEG was recorded with a 10–20 system during centers for several years. The participants had additional the sleeping and waking state, and the infants received seizures after the first seizure or somewhat concerning fac- intermittent photic stimulation at time periods ranging tors. In addition, the collaborative hospitals were medi- from two months to one year and four months. All patients cal center hospitals with advanced medical technology to had EEG data available before one year of age except one treat refractory cases. Therefore, refractory cases tended to patient.

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SCN1A mutations were analyzed by the previously complex on EEG while the remaining 26 patients had only reported methods (Ohmori et al., 2002). In brief, ge- segmental myoclonus. Twenty-two of the 50 patients in the nomic DNA was extracted from peripheral blood cells. non-Dravet syndrome group experienced only FS. The re- Twenty-six exons of the SCN1A gene were amplified maining 28 patients had also experienced afebrile seizures. with the intronic primers. All PCR products were pu- We investigated the cutoff values of the age at onset of rified with a PCR products presequencing kit (Amer- a febrile seizure and the total number of seizures. Based sham Biosciences, Little Chalfont, Buckinghamshire, on the distribution of the age at onset of a febrile seizure United Kingdom), reacted with the Big Dye Termina- (Fig. 1A) and the ROC curve (Fig. 1B), seven months and tor FS ready-reaction kit (Applied Biosystems, Foster younger was selected as the cutoff point. This cutoff value City, CA, U.S.A.), and analyzed on an ABI PRISM3100 had a sensitivity of 0.94, a specificity of 0.66, a positive sequencer (Applied Biosystems). We performed a multi- predictive value of 0.72, and a negative predictive value plex ligation-dependent probe amplification (MLPA) us- of 0.92. Considering the distribution of the total number ing SALSA MPLA P137 SCN1A reagent for all SCN1A- of seizures (Fig. 1C) and the ROC curve (Fig. 1D), a to- mutation-negative patients to detect microdeletion (MRC- tal number of 5 and above was selected as the cutoff point. Holland, Amsterdam, The Netherlands). Twenty-nine of This cutoff value had a sensitivity of 0.89, a specificity of the 46 patients with Dravet syndrome had been previ- 0.84, a positive predictive value of 0.84, and a negative pre- ously enrolled in a SCN1A mutation analysis in our study dictive value of 0.89. (Ohmori et al., 2002). The patients’ characteristics before one year of age are summarized in Table 2. The Dravet syndrome group had Statistical analysis a significantly higher frequency of an age at onset ≤ The cutoff values of age at onset and the total number 7 months, a total number of seizures ≥ 5, hot water– of seizures were evaluated by the receiver operating char- induced seizures, and the presence of hemiconvulsions, fo- acteristic (ROC) curve. The sensitivity, specificity, positive cal seizures, myoclonic seizures, and prolonged seizures predictive value, and negative predictive value were calcu- lasting more than 10 min. Sex and family history were not lated by the differentiation of the participants who devel- significantly associated with Dravet syndrome. oped Dravet syndrome and those who did not. The ROC Among the patients who experienced prolonged seizures curves were plotted and the area under the curves (AUC) for more than 10 min, two patients in the non-Dravet syn- was calculated. drome group and 27 patients in Dravet syndrome group Chi-square or Fisher’s exact tests were used to test dif- (59%) had lasting more than 30 min. ferences for each potential predictive factor for Dravet The sensitivity of prolonged seizures for more than 10 min syndrome. Statistical modeling using logistic regression analysis was used to calculate the odds ratio (OR) of in- dividuals with Dravet syndrome to those without Dravet syndrome. ORs were expressed with the 95% confidence Table 1. Clinical characteristics of the interval (95% CI). We subsequently assigned a risk score participants at study enrollment for each predictive factor based on OR or p-value. If the Non- odds ratio ≤ 1orp> 0.01, the risk score = 0; if 1< OR Dravet Dravet − < 20 or 0.01 ≥ p >1 × 10 5, the risk score = 1; if 20 ≤ syndrome, syndrome, OR < 40 or 1.0 × 10−5 ≥ p > 1.0 × 10−10, the risk score Characteristics n = 50 n = 46 = 2; and if 40 ≤ OR or 1 × 10−10 ≥ p, the risk score = Mean age ± SD, (years) 11.6 ± 5.9 17.1 ± 8.4 3. We applied the risk score to each patient and calculated Subclassification Febrile seizures 22 (44%) 0 the final sum of the risk scores. The ability of the risk score Febrile seizures + afebrile CPS 10 (20%) 0 screening test to discriminate Dravet syndrome from non- Febrile seizures + afebrile CPS, GTC 4 (8%) 0 Dravet syndrome was evaluated using ROC curve analysis. Febrile seizures + afebrile GTC 9 (18%) 0 ROC curves were also used to compare the diagnostic per- Febrile seizures + afebrile GTC, MyS 1 (2%) 0 formance of different sets of risk factors. BCECS 2 (4%) 0 GEFS+ 1(2%) 0 All statistical analysis in this study was performed us- Childhood absence epilepsy 1 (2%) 0 ing the SPSS software package version 12.0 (SPSS Inc., Mental development Tokyo, Japan). Normal (IQ ≥ 70) 45 (90%) 0 Mild retardation (50 ≤ IQ<70) 5 (10%) 4 (9%) Moderate to severe retardation (IQ < 50) 0 42 (91%) RESULTS SD, standard deviation; PS, complex partial seizure; The clinical characteristics of the participants are sum- GTC, generalized tonic–clonic seizure; MyS, myoclonic seizure; marized in Table 1. In the Dravet syndrome group, 20 of the BCECS, benign childhood epilepsy with centrotemporal spikes; + 46 patients had myoclonic seizures with spike-and-wave GEFS , generalized epilepsies with febrile seizures plus.

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Figure 1. Distribution of age at onset and number of seizures, and ROC curves. The cutoff values were evaluated by distribution of age at onset in patients (A), ROC curve of age at onset (B), distribution of number of seizures in patients (C), and ROC curve of number of seizures (D). Epilepsia C ILAE is higher than that of status epilepticus lasting more than had various types of mutations. Approximately half of 30 min. Our aim of this study is to develop a screening these mutations were missense and half were truncated mu- test in order to predict Dravet syndrome, therefore, we se- tations containing nonsense mutations and frameshift mu- lected the prolonged seizures of more than 10 min as a risk tations. A deletion of exon 10 was detected in one patient factor. using MPLA. SCN1A missense and truncated mutations The photoparoxysmal response (PPR) on EEG was ob- were detected significantly more often in the Dravet syn- served in 16 of the 46 patients (32%) in the Dravet syn- drome group than in the non-Dravet syndrome group. Clin- drome group during the clinical course. These 16 patients ical characteristics of SCN1A-mutation-positive patients in belonged to the typical Dravet syndrome group (20 pa- non-Dravet syndrome group were shown in the supplemen- tients). PPR appeared before one year of age in one patient, tary Table 2. between one and two years of age in four patients, between Risk scores for each factor were assigned to predict two and three years of age in four patients, and after three Dravet syndrome based on their ORs and p-values as de- years of age in seven patients. PPR on EEG before one year scribed in subjects and methods (Table 3). We applied of age was not useful for predicting Dravet syndrome in our the risk score to each patient’s clinical risk factors at one patients. year of age and calculated the sum. The ROC curves were The SCN1A mutations in the participants were summa- used to establish the cutoff points for each screening test rized in the supplementary Table 1. Six of the 50 patients (Table 4). The cutoff point of the total clinical score to iden- (12.0%) in the non-Dravet syndrome group had SCN1A tify Dravet syndrome was established as 6 or above (Model mutations. All of these were missense mutations. However, 3). If hot water–induced seizures were not observed be- 38 of the 46 patients (82.6%) in the Dravet syndrome group cause the patient did not bathe in hot water, this factor

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Table 2. Patients’ characteristics before one year of age

Non-Dravet, n = 50 Dravet, n = 46 p-value Odds ratio 95%CI Patients’ characteristics at onset Mean age at onset (months) 8.2 5.2 Onset ≤ 7 months 17 (34%) 43 (93%) 6.62 × 10−10∗ 27.82 7.52–102.97 Sex, male 25 (50%) 20 (43%) 0.522 1.3 0.58–2.91 Family history in first-degree relatives 25 (50%) 13 (28%) 0.03 0.39 0.17–0.92 Family history in second-degree relatives 31 (62%) 19 (41%) 0.043 0.43 0.19–0.98 Number of seizures before one year of age Total number ≥ 5 8 (16%) 41 (89%) 1.23 × 10−13∗ 43.05 13.00–142.55 before one year of age GTC, GC 50 (100%) 44 (96%) 0.227∗ 0 Hemiconvulsion 1 (2%) 33 (72%) 9.42 × 10−14∗ 124.39 15.52–996.92 2 (4%) 17 (37%) 5.61 × 10−5∗ 14.07 3.03–65.36 Myoclonic seizure 0 (0%) 6 (13%) 0.01∗ ∞ – Prolonged seizure 3 (6%) 37 (80%) 1.38 × 10−14 64.41 16.27–254.94 Precipitating stimuli Hot water 3 (6%) 27 (59%) 1.28 × 10−8∗ 22.26 6.03–82.22 Laboratory characteristics Epileptic discharges on EEG 13 (26%) 4 (9%) 0.033∗ 0.27 0.08–0.90 Genetic test SCN1A missense mutation 6 (12%) 19 (41%) 0.001 5.16 1.83–4.53 SCN1A truncated mutation 0 (0%) 20 (43%) 2.59 × 10−8∗ ∞ – ∗p-value by Fisher’s extract test. should be deleted. In this case, the cutoff point was 5 and dependent, a definitive diagnosis based on the presence of above (Model 4). The cutoff points for the sum of clini- all the symptoms is delayed until early childhood. It is dif- cal and genetic score were also calculated in the same way ficult to differentiate Dravet syndrome from benign febrile (Models 1 and 2). Seven and above for the cutoff point before an infant’s first birthday. Both of these of the model 1 exhibited high sensitivity and specificity. disorders manifest as febrile convulsions with no neurolog- Figure 2 illustrates the distribution of the sum of the ical delay at the age of onset. Furthermore, general pedia- risk scores when each model is applied to an individual. tricians who treat FS in infants have difficulty in discrim- Table 4 summarizes the predictive value of each model for inating between Dravet syndrome and benign FS. Dravet sensitivity, specificity, PPV, and NPV. syndrome is characterized by recurrent seizures and sta- tus epilepticus during infancy, so it is necessary for a child DISCUSSION neurologist or to treat patients with this syn- drome as early as possible. We therefore sought to develop Dravet syndrome is a refractory epileptic syndrome dis- a risk score screening test to predict Dravet syndrome be- playing various types of seizures. Due to the fact that the fore one year of age. appearance of the clinical features of the syndrome is age Regarding the characteristics of infants at the onset of Dravet syndrome, there were no data on how often Dravet syndrome patients have seizures before one year Table 3. A proposed risk score for a screening of age. Our data suggested that a total number of five or test more seizures was the cutoff value in distinguishing be- Predictive risk factors Risk score tween Dravet syndrome and non-Dravet syndrome. Re- garding the characteristic seizure types, the incidence rates Clinical score Onset ≤ 7 months 2 of hemiconvulsions, focal seizures, myoclonic seizures, Total number of seizures ≥ 53and prolonged seizures lasting more than 10 min were Hemiconvulsion 3 significantly higher in Dravet syndrome. FS in Dravet Focal seizure 1 syndrome often eventually evolve into status epilepticus. Myoclonic seizure 1 Oguni et al. (2005) reported that the incidence of status Prolonged seizure 3 Hot water–induced seizure 2 epilepticus was 67–77%. In our series, status epilepticus Genetic score was observed in 27 patients (59%) before the first year of SCN1A missense mutation 1 life. The rate of status epilepticus was thus lower in our SCN1A truncated mutation 2 study than in the previous study. A family history of con- vulsive disorders (including FS and epilepsy) was reported

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Table 4. Diagnostic value of combination of risk factors

Screening test Non-Dravet, Dravet, (No. of risk factors) n = 50 n = 46 Sensitivity Specificity PPV NPV Model 1 (9 factors) Clinical score + genetic score ≥ 7 1 45 0.978 0.980 0.978 0.980 Model 2 (8 factors) Clinical score − hot water + genetic score ≥ 6 5 44 0.957 0.900 0.989 0.957 Model 3 (7 factors) Clinical score ≥ 6 3 45 0.978 0.940 0.938 0.979 Model 4 (6 factors) Clinical score − hot water ≥ 5 6 44 0.957 0.880 0.880 0.957 Model 5 (2 factors) Genetic score≥ 1 6 38 0.826 0.880 0.864 0.846 PPV, positive predictive value; NPV, negative predictive value. in 25–64% of patients with Dravet syndrome (Dravet et al., Hot water–induced seizures are often observed in 2005). In this study, both the Dravet syndrome group and Japanese patients with Dravet syndrome (Oguni et al., the non-Dravet syndrome group had a high frequency of a 2001; Ohmori et al., 2003). The Japanese have a custom of family history of seizures, so there was no significant dif- bathing with hot water at 40–42◦C. Most of the patients had ference between the groups. experienced a generalized or unilateral after el- Risk factors for epilepsy after FS have been inves- evation of the body temperature by a hot bath for several tigated for a number of years. An onset of seizures minutes. A significantly higher ratio of this phenomenon before one year of age, convulsions with focal fea- in patients with Dravet syndrome may suggest that Dravet tures, prolonged seizures, repeated seizures within 24 h, syndrome displays a greater degree of thermosensitivity and a family history of seizures in first-degree relatives than common FS. have been reported as risk factors (Nelson & Ellenberg, The incidence of epileptic discharge on EEG in the non- 1976; Annegers et al., 1979; Annegers et al., 1987). It Dravet syndrome group was high (26%). Twenty-eight of is interesting that the risk factors for Dravet syndrome the 50 patients (56%) in the non-Dravet syndrome group were similar to those for epilepsy after FS in some had subsequent afebrile seizures. The non-Dravet syn- respects. drome group included not only benign cases with simple

Figure 2. Distribution of the sum of the risk scores when the Model 1 is applied to each patient. Epilepsia C ILAE

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FS but also the complicated cases. As a result, the com- tance. Since the parental mosaicism of SCN1A mutations plicated cases in the non-Dravet syndrome group may thus can lead to sibling cases of Dravet syndrome, an SCN1A contribute to high rates of epileptic discharges and subse- mutation analysis will thus become more beneficial for ge- quent epilepsy. netic counseling rather than a diagnostic test of Dravet syn- Early photosensitivity is known to be one of the EEG drome (Gennaro et al., 2006; Marini et al., 2006; Morimoto characteristics of Dravet syndrome. Our subjects included et al., 2006). both typical Dravet syndrome and borderline Dravet syn- FS commonly begin between five months to six years drome. The incidence of photosensitivity in borderline of age. The occurrence of afebrile seizures after a febrile Dravet syndrome cases is usually low (Oguni et al., 2001; seizure is 2–7% (Nelson & Ellenberg, 1976, Annegers Ohmori et al., 2003). This may therefore be the cause of the et al., 1979, Annegers et al., 1987). In this study, 28 of lower incidence of PPR on EEG in our cases in comparison the 50 patients (56%) with a febrile seizure before one to previous studies (Dravet et al., 2005). year of age later experienced afebrile seizures. The patients SCN1A mutations were initially found in large families in the non-Dravet syndrome group also needed careful with GEFS+ in 2000 (Escayg, 2000). A missense SCN1A observation; however, they had a much more favorable mutation was reported in a family with children who ex- outcome compared with those in the Dravet syndrome perienced simple febrile convulsions (Mantegazza et al., group. 2005), but this mutation was considered to be a rare case As a result of our findings, we would like to propose the (Malacarne et al., 2002). There was no comparison of the practical use of the screening test. If the sum of clinical risk detection of SCN1A mutations in children under one year scores ≥ 6, the patient has a high risk of Dravet syndrome of age with FS who did and did not develop Dravet syn- (Model 3). If the patient does not habitually bathe in hot drome. In this study, 6 of the 50 (12%) patients who expe- water, a sum of the clinical risk scores ≥ 5 is recommended rienced FS before their first birthday had SCN1A missense as the predictive value for Dravet syndrome (Model 4). The mutations. The rate of SCN1A mutations in the non-Dravet SCN1A mutation analysis may be not available for all pa- syndrome group was higher than that reported in previous tients due to the high expense of the test. If the sum of studies (Escayg et al., 2001; Wallace et al., 2001). In pre- the clinical risk scores before a genetic test is conducted is vious studies, the subjects included idiopathic generalized ≥ 6, the SCN1A mutation analysis is recommended. We fo- epilepsy such as juvenile , childhood cused on the utilization of the risk score screening test by absence epilepsy, juvenile absence epilepsy, and general- general pediatricians. The proposed risk score could help to ized tonic–clonic seizures on awakening. A single-stranded identify Dravet syndrome among infants who experience a conformation analysis was undertaken for mutation screen- febrile seizure. This proposal is a novel approach to pre- ing. The difference in the detection rate might be due to dict Dravet syndrome before one year of age. Since it is a differences in either the subjects or in the methodology of case control study, our proposal should be prospectively the genetic test. validated and further improved to increase its predictive Our data exhibited that both missense mutations and value. truncated mutations were significantly more common in the Dravet syndrome group. The SCN1A mutation analysis ACKNOWLEDGMENTS was a potential test for the prediction of Dravet syndrome. We are grateful to Dr. AL George Jr. for critical reading of the Dravet syndrome has been diagnosed by a comprehen- manuscript. We also thank Drs. Y. Maegaki, K. Ogawa, R. Amano, M. sive evaluation of seizure types, clinical course, and EEG Fujita, K. Kobayashi, and N. Murakami who helped to collect clinical findings. An accurate diagnosis of Dravet syndrome at an data and blood samples. We confirm that we have read the Journal’s posi- tion on issues involved in ethical publication and affirm that this report is early age requires an experienced child neurologist. Our consistent with those guidelines. risk score screening test in this study may enable general pediatricians to identify patients with Dravet syndrome at Disclosure of Conflicts of interest We have no conflicts of interest. an early stage. A clinical risk score sum ≥ 6 was a suc- cessful cutoff value with a high sensitivity and specificity (Table 4, Model 3). Although adding a genetic score im- REFERENCES proved the diagnostic value of specificity, PPV, and NPV Annegers JF, Hauser WA, Elveback LR, Kurland LT. (1979) The risk of (Model 1), clinical score seemed to have sufficient sensitiv- epilepsy following febrile convulsions. Neurology 29:297–303. ity and specificity (Model 3). Since the SCN1A mutations Annegers JF, Hauser WA, Shirts SB, Kurland LT. (1987) Factors prog- were detected in other childhood epileptic syndromes, the nostic of unprovoked seizures after febrile convulsions. N Engl J Med 316:493–498. SCN1A-associated epilepsies are considered to have a wide Claes L, Del-Favero J, Ceulemans B, Lagae L, Van Broeckhoven C, De spectrum (Harkin et al., 2007). Moreover, considering that Jonghe P. 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Screening Test for Dravet Syndrome

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Epilepsia, 49(4):626–633, 2008 doi: 10.1111/j.1528-1167.2007.01475.x