Clinical and genetic familial study of a large cohort of Italian children with idiopathic epilepsy

Brain Research Bulletin 79 (2009) 89–96

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Clinical and genetic familial study of a large cohort of Italian children with idiopathic epilepsy Romina Combi a , Daniele Grioni b , Margherita Contri b , Serena Redaelli c , Francesca Redaelli d , Maria Teresa Bassi d , Donatella Barisani e , Maria Luisa Lavitrano f , Giovanni Tredici c , Maria Luisa Tenchini g , Mario Bertolini b,c , Leda Dalprà c,∗ a

Department of Biotechnology and Biosciences, University of Milan-Bicocca, p.zza della Scienza 2, 20126 Milano, Italy Infantile Neuropsychiatry Clinic, S Gerardo Hospital, via GB Pergolesi 33, 20052 Monza, Italy c Department of Neurosciences, University of Milan-Bicocca, via Cadore 48, 20052 Monza, Italy d E Medea Scientific Institute, Laboratory of Molecular Biology, via Don Luigi Monza 20, 23847 Bosisio Parini (LC), Italy e Department of Experimental Medicine, University of Milan-Bicocca, via Cadore 48, 20052 Monza, Italy f Department of Surgical Sciences and Intensive Therapy, University of Milan-Bicocca, via Cadore 48, 20052 Monza, Italy g Department of Biology and Genetics for Medical Sciences, University of Milan, via Viotti 3, 20133 Milano, Italy b

a r t i c l e

i n f o

Article history: Received 11 April 2008 Received in revised form 19 December 2008 Accepted 16 January 2009 Available online 5 February 2009 Keywords: Epilepsy Mutation Ion channels Genetics Cohort

a b s t r a c t Epilepsies are characterized by genetic heterogeneity and by the possible coexistence of different phenotypes in one family. Moreover, in different epilepsies, mutations in the same gene have been reported. We aimed to collect data in a large Italian cohort of 81 families with children affected by partial or generalized epilepsies and to evaluate the prevalence of several ion channel mutations. In particular, a clinical and genetic survey was performed and DNA regions known to be associated with several epilepsies were analysed by sequencing. We observed genetic complexity in all phenotype groups: any epileptic type may be transmitted as either autosomal dominant or recessive. No significant phenotype identity among generations and no differences among genders could be observed. Two missense mutations in SCN1A were identified in two GEFS+ probands confirming the importance of this channel for this epilepsy. Moreover, a previously unreported CLCN2 mutation was detected in a proband showing CAE. In conclusion, even in this highly heterogeneous cohort, the complexity of the epileptic condition was highlighted and mutations in the analysed candidate region of ion channel genes appear to explain only a minority of cases. © 2009 Elsevier Inc. All rights reserved.

1. Introduction Epilepsies are common, heterogeneous neurological disorders in which both genetic and environmental factors play a role. Idiopathic epilepsies are assumed to have a strong genetic component, being monogenic or oligo/polygenic with different recurrence risks in the same family [20]. However, even in monogenic epilepsy, additional genes and environmental factors may modulate its expression, thus resulting in incomplete penetrance and variable phenotype. Despite these difficulties, during the last 10 years several gene loci have been mapped by means of linkage analysis, and mutations have been detected in genes encoding ion-channels, leading

∗ Corresponding author at: Department of Neurosciences and Biomedical Technologies, University of Milan-Bicocca, Via Cadore 48, 20052 Monza, Italy. Tel.: +39 02 64488300; fax: +39 02 64488250. E-mail address: [email protected] (L. Dalprà). 0361-9230/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2009.01.008

to hyperexcitability of cortical neurons through alterations in the channel function [20], as well as in genes not belonging to the channel family [33]. Mutations in potassium, sodium and chloride channels as well as in acetylcholine and GABA receptors have been identified [16]. Therefore, seizures could be induced either by the enhancement of the excitatory stimuli or by impairment of the inhibitory mechanisms [35]. Interestingly, mutations in SCN1A and SCN2A encoding the ␣-1 and ␣-2 subunits of the sodium channel have been identified in patients with GEFS+ epilepsy, but also in SMEI or Dravet syndrome, a severe myoclonic epilepsy of infancy [1,14,19,22,26,27,31]. However, GEFS+ is also associated with mutations in SCN1B, encoding the ␤-1 subunit of the sodium channel [32], and in the GABRG2 gene, encoding the ␥-2-subunit of a GABAA receptor involved in childhood absence epilepsy (CAE) with febrile seizures (FS) [2,4,18,21,30]. Given all the genetic findings in the field of the molecular pathophysiology of epilepsy, we collected 81 Italian families in order to study clinical and genetic data in a large cohort of Mediterranean origin. Moreover, we tested some of the known ion channel

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mutations associated with several epilepsies in 46 compliant probands. 2. Material and methods 2.1. Family recruitment, inclusion criteria and clinical evaluation Affected children with a familial history of epilepsy were selected from the database of all cases collected by the Epilepsy Unit of the Infantile Neuropsychiatric Department of San Gerardo Hospital in Monza, Italy. We considered as ‘familial epilepsy’ the occurrence of epilepsy in at least two members of the same family including the proband. Individuals with different forms of idiopathic generalized and focal epilepsy and individuals with febrile seizures were enrolled in the study. Patients showing symptomatic epilepsy were excluded. In all probands, a complete diagnostic evaluation was performed, inclusive of: (1) collection of a detailed medical history of epileptic disorder in the proband and relatives (pregnancy and delivery, type of seizures and age of onset, seizure susceptibility to fever, drug resistance); (2) complete neurological examination and neurophysiological studies; (3) review of all available medical records (neurological examination reports, pharmacological treatments, CT and MRI scans, EEG studies). For each proband the pedigree was extended as far back as possible. We collected the medical history and reviewed available medical records to define the epileptic syndrome in relatives; if necessary we performed new clinical examinations. 2.2. Epilepsy classification Epileptic seizures and syndromes were classified, whenever possible, according to the International League against Epilepsy criteria [8–13]. Patients were classified into the following epileptic syndromes: • • • • • • • • • • • • • • •

Early onset benign childhood occipital epilepsy panayiotopoulos type (BEOP). Benign childhood epilepsy with centrotemporal spikes (BCECTS). Benign familial neonatal seizures (BFNS). Benign familial infantile seizures (BFIS). Idiopathic generalized epilepsy: epilepsy with generalized tonic clonic seizures only (IGE). Juvenile myoclonic epilepsy (JME). Childhood absence epilepsy (CAE). Myoclonic epilepsy of infancy (MEI). Generalized epilepsy with febrile seizure plus (GEFS+). Epilepsy with myoclonic-astatic seizures of childhood (MAE). Severe myoclonic epilepsy in infancy (SMEI) or Dravet syndrome. Febrile seizures (FS). Benign focal seizures of adolescence (BFSA). Epileptic encephalopathy with continuous spike-and-wave during sleep (CSWS). Familial temporal lobe epilepsy (FTLE).

Patients who changed epilepsy phenotype during their life course were classified according to their epileptic syndrome at onset (changes are reported in Supplementary Table 1). Some epileptic syndromes could not be easily classified according to international classification. We attributed to “benign focal seizures of adolescence” (BFSA) [5] cases in which seizures occurred in young subjects, starting in their second decade of life, and which were characterized by focal seizures with or without secondary generalization. The interictal EEG, neurological examination, and scans were normal and the epilepsy had a benign course during a mean follow-up of 3 years. 2.3. Polymerase chain reaction (PCR) And DNA sequencing Probands’ parents or probands themselves (>18 years old) signed an informed consent form and the study was approved by the Ethical Committee of S. Gerardo Hospital. Venous blood was collected in 0.125 M trisodium citrate, and genomic DNA was extracted using the Wizard genomic DNA purification kit (Promega, Madison, WI, USA). PCRs were performed on 100 ng of genomic DNA in a 25 ␮L volume. The reaction mixture contained 1 × reaction buffer (10 mM Tris [tris (hydroxymethyl) aminomethane]–HCl, pH 8.3, 50 mM KCl, 0.01% gelatin), 1.5 mM MgCl2 , 10 ␮M of each primer, 100 ␮M dNTPs and 1.5 U REDTaq DNA polymerase (Sigma, St. Louis, MO, USA). PCRs were carried out on an Ampligene-9700 thermal cycler (Applied Biosystems, Foster City, CA, USA) under standard conditions. Primers (Life Technologies, Inchinnan, Paisley, UK) were designed on the basis of the known genomic sequence of each gene using Oligo 4.0 software. In particular, all probands underwent a screening study aimed at finding mutations in 8 genes covering 100% of the reported mutations in CACNA1A, CACNB4, GABRA1 and SCN1B, 55% in CLCN2, 80% in GABRG2, 43% in SCN1A and 23% in SCN2A. The analyses were limited to the gene regions whose involvement was reported more frequently. This was because, during the study, we found so few mutations that we were forced to conclude that our intention to sequence all the coding sequences was impracticable and, moreover, more expensive and time-consuming than expected. The analysed mutations are listed in Supplementary Table 2 together with the primers used in the study.

Owing to the fact that the SCN1A gene is frequently mutated in patients affected by GEFS+ or SMEI, an additional study was performed whereby a complete sequencing of the coding parts of this gene was carried out in these patients. The specific PCR conditions for each primer couple and the sequence of all primers used for SCN1A sequencing are available on request. In the event of exonic mutations, primers were designed to amplify the flanking intronic regions, allowing the sequencing of the whole exon and exon/intron boundaries whereas, for intronic mutations, primers were designed to amplify a region of approximately 500 bp encompassing the mutation. Sequencing was carried out directly on purified PCR products. Sequence analysis was carried out on both strands using the BigDye Terminator Cycle Sequencing kit v3.1 and an automated ABI-3100 DNA sequencer (Applied Biosystems). Where a nucleotide variation was identified in a patient, its presence was tested in all available DNAs from the relatives. 2.4. Bioinformatic tools and statistical analysis Factura and Sequence Navigator software packages (Applied Biosystems) were used for mutation detection. The existence of detected nucleotide variations was verified by comparing them with the NCBI (http://www.ncbi.nlm.nih.gov/) and Ensembl databases (http://www.ensembl.org/). The presence of possible common genotypes or haplotypes was determined using Phase 2.1 Software [25], whereas an in silico analysis of the possible effects of the detected nucleotide variations was performed by employing SpliceView and HMMGene software for alterations in splicing sites (http://l25.itba.mi.cnr. it/∼webgene/wwwspliceview.html; http://www.cbs.dtu.dk/services/HMMgene/), ESEfinder for alterations in splicing enhancers (http://rulai.cshl.edu/tools/ESE/ index.html) and PolyPhen software for the prediction of possible mutation effects (http://coot.embl.de/PolyPhen/) [29]. The Chi-squared test with the appropriate corrections was used to evaluate the presence of statistical differences among selected groups. Differences were considered significant when p < 0.05.

3. Results 3.1. Family characteristics We selected 81 families with two or more members affected by different forms of both generalized and focal idiopathic epilepsies. The mean number of persons (both affected and healthy) per family was 9.5 with a mean of 2.7 affected individuals. Families were classified according to the proband’s epilepsy syndrome. The total number of subjects was 748, of whom 214 (28.2%) showed epilepsy. There were 83 probands owing to the fact that families 41 and 43 were recruited through 2 probands for each family and were found to be first cousins. Probands were equally distributed between the sexes: 40 females (48%) and 43 males (52%). Among parents only 40 were found to be affected (24 fathers and 16 mothers), representing 18.7% of the total of affected subjects. In 3 families both parents were affected. In 16 families an affected sibling was observed, with 9 brothers and 9 sisters in total (excluding probands). Moreover, the type of epilepsy was concordant in 8 of these siblings and, among them, 7 were concordant also for gender. Six families showed epilepsies in both paternal and maternal familial branches and, also in these cases, they were considered to be a unique family. Fifty-two first- and 24 second-degree relatives had epilepsy (42 males and 34 females). In the collected sample, only three sets of dizygotic twins were present, and in all cases the twins were discordant for epilepsy. 3.2. Sample composition and clinical data Probands’ clinical data are reported in Supplementary Table 1. The mean age of probands at the onset of epilepsy was 5.5 years (first week of life–15 years) and 10.7 years at follow-up (1–25 years). The follow-up duration ranged from 6 months to 23 years. Neuroradiological studies (CT scan or T1W, T2W, T2 FLAIR MRI) were normal in all cases. Neurological examination and psychomotor development were normal in all patients, except in patients with epileptic encephalopathies such as SMEI and MAE, who developed cognitive impairment and movement disorders such as ataxia or

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Fig. 1. Pedigrees of families in which the molecular analysis was performed. Families with the complete sequencing of the SCN1A of the proband are indicated with an asterisk. BEOP: early onset benign childhood occipital epilepsy (Panayiotopoulos type); MEI: myoclonic epilepsy of infancy; BCECTS: benign childhood epilepsy with centrotemporal spikes; GEFS+: generalized epilepsy with febrile seizures plus; FS: febrile seizures; MAE: epilepsy with myoclonic-astatic seizures of childhood; IGE: idiopathic generalized epilepsy: epilepsy with generalized tonic clonic seizures only; SMEI: severe myoclonic epilepsy or Dravet syndrome; JME: juvenile myoclonic epilepsy; BFSA: benign focal seizures of adolescence; CAE: childhood absence epilepsy; BFIS: benign familial infantile seizures.

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Fig. 1. (Continued ).

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pyramidal signs. In these cases, a high resolution karyotype and a subtelomeric region analysis by FISH with specific probes were performed, and these resulted normal. Twenty-two patients (26.5%) did not undergo pharmacological treatment whereas 61 patients (73.5%) took antiepileptic drugs; among these, 18 patients (29.5%) discontinued therapy after a mean period of 4.2 years. Patients with benign focal epilepsy of infancy (i.e. BEOP, BCECTS) and febrile seizures underwent therapy only in selected cases. All patients are seizure-free at present, except for two cases of MAE (62-III-1 and 63-III-1) and one case of SMEI (64-IV-2); these patients suffer from drug-resistant seizures. 3.3. Genetic analysis With regard to epileptic features in general, pedigrees were compatible with an autosomal dominant transmission (AD) with an incomplete penetrance which is very variable among families, the overall penetrance being 67%. In 43 families an autosomal recessive (AR) transmission could also be hypothesized. In particular, with regard to the IGE, CAE and GEFS+ phenotypes, 19 (57%) out of 35 families could be compatible with this transmission, while considering the remaining phenotypes all together this range is lower (about 53%). We observed the same epilepsy phenotype in the affected child and parent in 14 families: 2 BCECTS; 5 IGE; 1 CAE; 2 GEFS+; 1 BFSA; 2 FS (in one of them both parents were affected). On the other hand, in family 37 the proband was affected by JME, the mother and the first child both showed FS, whereas the father and his sister were affected by BFSA (Fig. 1); in this family a double transmission could be hypothesized. Affected subjects could be distributed into 3 main groups, as shown in Fig. 2: the proband group appeared more variegated as compared to the others but no significant differences were detected (2 test). IGE was the most represented form of epilepsy in each group, followed by FS and CAE. The group of relatives included individuals belonging to three generations. In term of transmission probability, no significant correlations were observed between the phenotype of parents and children: parents with focal/generalized epilepsy could have children with either generalized or focal disease. 3.4. Mutational screening The presence of several already reported mutations linked to epilepsy was verified in 46 probands affected by different epileptic forms (Fig. 1). The probands of the remaining 35 families were not considered in this part of the study because their genetic material was not available. Among the mutated regions, one (Arg19Lys in SCN2A exon 2), previously reported in GEFS+ patients and referred to as probably being a benign variant associated with susceptibility to epilepsy [26], was detected in a heterozygous state in six probands affected by BEOP (family 3), BCECTS (families 9 and 10), IGE (families 22 and 31) and GEFS+ (family 60). Sequencing allowed the detection of additional known and unknown nucleotide variations. In particular, we detected six new nucleotide variations (Table 1) and 9 already known SNPs distributed in three and four genes, respectively. With regard to the newly identified nucleotide variations, two out of six were located in intronic regions and were detected by the sequencing of intron/exon boundaries of the relevant exonic sequence. One was identified in 20 probands (9 in a heterozygous and 11 in a homozygous state) by the SCN1B exon 3 sequencing; a second one was identified in CLCN2 intron 2 in 9 probands (all heterozygous for this variation).

Fig. 2. Epilepsy phenotypes encountered in the different generations. Pies representing the proportion of each phenotype in different generations (probands, parents and relatives) are shown. BEOP: early onset benign childhood occipital epilepsy (Panayiotopoulos type); BCECTS: benign childhood epilepsy with centrotemporal spikes; BFIS: benign familial infantile seizures; IGE: idiopathic generalized epilepsy: epilepsy with generalized tonic clonic seizures only; JME: juvenile myoclonic epilepsy; CAE: childhood absence epilepsy; MEI: myoclonic epilepsy of infancy; GEFS+: generalized epilepsy with febrile seizures plus; MAE: epilepsy with myoclonic-astatic seizures of childhood; SMEI: severe myoclonic epilepsy or Dravet syndrome; BFSA: benign focal seizures of adolescence; FS: febrile seizures.

A new silent variation (Leu1561Leu) was detected in SCN1A exon 26. This variation (identified in one CAE proband in a heterozygous state) was a C->T transition which resulted in no amino acid change. An in silico analysis, performed by means of online software (i.e. SpliceView and HMMGene), revealed that this variation does not introduce or remove any splicing sites nor does it affect the exonic splicing enhancer (ESE) pattern (as detected by the ESEfinder). Two missense mutations were identified by the sequencing of the whole SCN1A performed in 9 probands affected by GEFS+ and SMEI (Table 1). The Arg542Gln mutation, which was previously associated with autism and reported in one sporadic case affected by JME [15,34], was detected in exon 10 in a GEFS+ patient (family 57, Fig. 1). The segregation analysis highlighted a de novo origin of this mutation, being absent in the two parents while several other polymorphisms, localized on 5 different chromosomes, were compatible with the parental transmission. This mutation disrupts the predicted tyrosine kinase site RLTYEKRY in the cytoplasmic loop 1,

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Table 1 New polymorphisms detected by sequencing. Gene

Localization

SCN1B SCN1A SCN1A SCN1A CLCN2 CLCN2

Intron 3 Exon 10 Exon 26 Exon 26 Intron 2 Exon 19

Nucleotide positiona 3722 29159 66962 68027 3354 9405

Nucleotide variation

Aminoacid variation

Reference sequence

Phenotype of the proband

T/C G/A C/T A/G G/A C/T

/ Arg542Gln Leu1561Leu Arg1916Gly / Ser719Leu

ENSG00000105711 ENSG00000144285 ENSG00000144285 ENSG00000144285 ENSG00000114859 ENSG00000114859

GEFS+, CAE, IGE, BCECTS, SMEI, JME GEFS+ CAE GEFS+ IGE, BCECTS, GEFS+ CAE/IGE

BCECTS: benign childhood epilepsy with centrotemporal spikes; IGE: epilepsy with generalized tonic clonic seizures only; JME: juvenile myoclonic epilepsy; CAE: childhood absence epilepsy; SMEI: severe myoclonic epilepsy or Dravet syndrome; GEFS+: generalized epilepsy with febrile seizures plus. a Nucleotide positions refer to the ENSEMBL Genomic sequence.

thus suggesting a possible effect on the regulation mechanism of the channel activity. The Arg1916Gly mutation was identified in heterozygosis in exon 26 of SCN1A in one GEFS+ proband (family 61, Fig. 1) (Human Mutation Database #Hm0635) and it was absent in 100 unrelated control individuals. Sequencing of the patient’s parents demonstrated a cosegregation of this variation with the GEFS+ phenotype, being present in heterozygosis in the affected father and absent in the mother. An in silico analysis performed with the online Polyphen software, which predicts the functional effects of human cnSNPs, suggested a probably damaging effect of the amino acid change. In fact, the Arg1916 is highly conserved among the alpha-subunits of voltage-gated sodium channels [7]. Finally, a new missense (Ser719Leu) mutation was detected in CLCN2 exon 19 in the proband of family 47 and it was absent in 100 unrelated control individuals. This proband showed CAE. A segregational analysis demonstrated that the mutation was transmitted by the mother who is a healthy carrier. The EEG studies performed on the mother showed bursts of phantom sharps and waves during hyperventilation. Unfortunately, the DNA of the cousin was not available for genetic testing. No particular allelotype/s and genotype/s were observed using the Phase2.0 software since all the possible combinations were randomly present. No significant differences were detected by comparing the allelic frequency of three known polymorphisms in our sample with literature data. In family 7 (Fig. 1) Neurofibromatosis type 1 (NF1) was also present, but the segregation of the epilepsy phenotype and NF1 was observed to be discordant. The linkage study with NF1 gene microsatellites revealed the same haplotype in subjects affected by neurofibromatosis (I2; II3; III2), whereas the BCECTS epilepsy was manifested in the father and first child unaffected by NF1. Because of a previous published linkage study [24] between BCECTS and alpha 7 subunit of neuronal nicotinic acetylcholine receptor, mapped on chromosome 15q13-14, we performed a segregation study by means of STR analysis and we observed that the epileptic children received different chromosome 15 s from the affected father (data not shown). 4. Discussion In order to provide further data leading to a better understanding of epilepsy genetics, we studied a large Italian cohort of affected families by analysing their clinical characteristics, the mode of transmission and the presence of some mutations in known genes described as being associated with epilepsy. Partial epilepsies represented 28% of the analysed sample, whereas the remaining 72% was constituted by idiopathic generalized epilepsies. From a genetic point of view, we observed a complexity in all phenotype groups since the transmission could be both autosomal dominant or recessive. No significant phenotype identity among generations could be observed considering the whole sample; in fact few affected parents and children shared the same phenotype,

with the exception of IGE families in which a phenotype concordance of 72% among probands and first and second degree relatives was observed. The distribution of the affected subjects was similar in the two sexes and no difference among all the transmitting parents (both penetrant and non-penetrant, irrespective of seizure type) was observed when the gender of the carrier was considered for each syndrome. Moreover, a pure mitochondrial maternal inheritance can be rejected as a transmission pattern, since a fraction of maleto-male transmission exists. By comparing the epilepsy phenotypes encountered in each analysed group (probands, parents, relatives) a difference, although not significant, could be observed (Fig. 2), since an increased number of phenotypes was detected in the proband group. This difference could be explained by the better diagnostic ability that has been acquired in the last few decades. Moreover, the presence of a large group of non-penetrant parents must be highlighted, since it could be an additional factor contributing to the minor variability observed in parents. Finally, it must be remembered that, in the relatives group, grandparents are included, a generation characterized by old-fashioned diagnoses and insufficient clinical documentation. Apart from the identification of the type of transmission, various studies have tried to assess whether the presence of an epilepsy phenotype is due to mutations in genes codifying for ion channels. In a mutation analysis of Nav 1.2 genes in 19 unrelated Japanese families with patients affected by GEFS+ or febrile seizures associated with afebrile seizures [26], an Arg19Lys change was detected in 5 out of 19 families and in 9 out of 112 controls. These observations led to the variant being defined as benign, although the high percentage of epileptic patients (5/19 vs 9/112, p = 0.0424) suggests that its role is more probably that of a modifier. In our sample, this polymorphism was present in 6 probands out of the 46 tested (13%) that showed different types of epilepsy: BCECTS (2×), BEOP, IGE (2×) and GEFS+, thereby suggesting a minor role for this variant in the pathogenesis of epilepsy. Two mutations were detected in the SCN1A. One was a missense mutation of paternal inheritance identified in a child showing febrile seizures at onset, followed by epileptic seizures (Fig. 1: family 61). The corresponding amino acid change appeared to be damaging for the protein function when analysed with the online Polyphen tool. However functional studies are necessary to determine whether this variation could have a pathogenetic role. The second mutation was detected in a child affected by GEFS+ (Fig. 1: family 57). The segregational analysis demonstrated that both parents were negative for this mutation suggesting a de novo origin. Interestingly, the father was affected by IGE as well as a first cousin. These data introduce two different interpretations owing to the absence of reports on a physiological effect of the mutation: the first is that the GEFS+ phenotype of the proband is due only to the identified mutation, while the father has a different epilepsy phenotype due perhaps to another mutation elsewhere which has been not transmitted to the proband; the second is that the introduction

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of a new mutation onto a previously established epileptic genetic background inherited from the father could have caused a partial change of the phenotype resulting in the manifestation of a different type of epilepsy. Several mutations have been reported in SCN1A, leading to the conclusion that this gene is responsible for the disease in about 13% of GEFS+ and 35-100% of SMEI patients [3,19]. Interestingly, these two phenotypes have been associated with mutations in other genes (for a review see [2]). On the other hand, the same subunits have been found to be mutated also in other epileptic syndromes [17], but how mutations in a single gene could cause mild (GEFS+) as well as severe (SMEI) phenotypes remains unexplained. Probably, genetic and environmental modifiers alter the host response to SCN1A mutations and the clinical expression of the mutation, as previously demonstrated in animal studies for the SCN8A gene [23]. In our cohort two out of 8 GEFS+ tested families carried a mutation (28%) in SCN1A, thus confirming the frequency reported in the literature. As far as SMEI is concerned, no mutations were detected in the only patient belonging to our cohort. Recently, three cases of SMEI in which a microdeletion encompassing the SCN1A gene was the molecular mechanism of origin have been reported, indicating that other mutations, apart from missense ones, are implicated in the pathogenesis [28]. However the frequency of mutations suggests that it may be worth searching for SCN1A mutations in all subjects with a diagnosis of GEFS+ and SMEI. No significant patterns of allelic combinations were detected, and taken all together the identified mutations in candidate regions of genes coding for channel or receptor subunits explain only a minority of cases. The analysed regions, chosen according to data reported in the literature, were recognized as harbouring mutations detected in subjects affected by different types of epilepsy covering, however, only a part of the coding sequence of the relevant genes. Therefore we cannot exclude the presence of mutations in the regions that did not undergo sequencing, taking into account that, as reported by several authors, no specific mutational “hot spot” can be detected. These observations suggest that new candidate genes could be searched for also in other gene families, as has been reported for ADNFLE, an epilepsy in which mutations in the candidate genes, coding for neuronal nicotinic acetylcholine receptor, have been detected in only a minority of cases and mutations in the corticotropin-releasing hormone gene have been found [6]. Thus the genetic complexity together with the clinical heterogeneity requires a multidisciplinary approach in order to obtain both a better clinical diagnosis and phenotype dissection.

Conflict of interest The authors declare that they have no competing financial interests.

Acknowledgements We thank all the study participants and in particular all children who agreed to the genetic test. We are grateful to prof. Luigi Ferini-Strambi for his valid suggestions on the analysis of the results.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.brainresbull.2009.01.008.

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