Epilepsy Research (2014) 108, 109—116
journal homepage: www.elsevier.com/locate/epilepsyres
Iterative phenotyping of 15q11.2, 15q13.3 and 16p13.11 microdeletion carriers in pediatric epilepsies Johanna A. Jähn a,1, Sarah von Spiczak a,b,1, Hiltrud Muhle a, Tanja Obermeier a, Andre Franke c, Heather C. Mefford d, Ulrich Stephani a,b, Ingo Helbig a,∗ a
Department of Neuropediatrics, University Medical Center Schleswig-Holstein (UKSH, Campus Kiel), Kiel, Germany b Northern German Epilepsy Center for Children and Adolescents, Schwentinental-Raisdorf, Germany c Institute of Clinical Molecular Biology, Christian-Albrechts-University Kiel, Kiel, Germany d Department of Pediatrics, Division of Genetic Medicine, University of Washington, Seattle, WA, USA Received 18 February 2013 ; received in revised form 16 September 2013; accepted 13 October 2013 Available online 26 October 2013
KEYWORDS Copy number variation; 15q11.2; 15q13.3; 16p13.11 microdeletions
Summary Microdeletions at 15q11.2, 15q13.3 and 16p13.11 are known genetic risk factors for idiopathic generalized epilepsies and other neurodevelopmental disorders. The full phenotypic range of this microdeletion triad in pediatric epilepsies is unknown. We attempted to describe associated phenotypes in a cohort of pediatric epilepsy patients. We screened 570 patients with pediatric epilepsies including idiopathic generalized epilepsies, focal epilepsies and fever-associated epilepsy syndromes for microdeletions at 15q11.2, 15q13.3 and 16p13.11 using quantitative polymerase chain reaction. Identified microdeletions were confirmed using array comparative hybridization. Ten microdeletions in 15q11.2 (n = 3), 15q13.3 (n = 3) and 16p13.11 (n = 4) were identified (1.8%). 9/10 microdeletions were identified in patients with IGE (6/101, 6.0%) or patients with generalized EEG patterns without seizures (3/122, 2.5%). 6/10 microdeletion carriers had various degrees of ID; the frequency of microdeletions in patients with epilepsy and ID was higher (4.6%) compared to patients with normal intellect (0.9%). Iterative phenotyping revealed a wide range of generalized epilepsy phenotypes. In our pediatric cohort, recurrent microdeletions at 15q11.2, 15q13.3 and 16p13.11 are mainly associated with phenotypes related to idiopathic generalized epilepsies or related EEG patterns. In contrast to
∗ Corresponding author at: Department of Neuropediatrics, University Medical Center Schleswig-Holstein (UKSH, Campus Kiel), ArnoldHeller-Str. 3, Haus 9, D-24105 Kiel, Germany. Tel.: +49 431 597 1761; fax: +49 431 597 1769. E-mail addresses: [email protected] (J.A. Jähn), [email protected] (S. von Spiczak), [email protected] (H. Muhle), [email protected] (T. Obermeier), [email protected] (A. Franke), [email protected] (H.C. Mefford), [email protected] (I. Helbig). 1 These authors have contributed equally.
0920-1211/$ — see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.eplepsyres.2013.10.001
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J.A. Jähn et al. previous reports, these recurrent microdeletions are virtually absent in focal epilepsies, FS, FS+ and GEFS+. Microdeletion carriers have a five-fold risk to present with various degrees of ID compared to patients without these risk factors. This microdeletion triad might help delineate a novel spectrum of epilepsy phenotypes classifiable through clinical, electrographic and genetic data. © 2013 Elsevier B.V. All rights reserved.
Introduction
qPCR
Microdeletions and microduplications are known genetic risk factors in common diseases including neuropsychiatric disorders such as schizophrenia, autism or intellectual disability. Rare and recurrent copy number variations (CNV) were also found to be associated with various epilepsy phenotypes. Microdeletions at 15q11.2, 15q13.3 and 16p13.11 are the most common CNVs in epilepsies occurring with a frequency of about 1,5% (Helbig et al., 2009; De Kovel et al., 2010). While microdeletions at 15q13.3 have only been described in association with idiopathic (genetic) generalized epilepsies (De Kovel et al., 2010), microdeletions at 15q11.2 and 16p13.11 were also found in generalized and focal epilepsy phenotypes (Heinzen et al., 2010). Notably, absence epilepsy in patients carrying a 15q13.3 microdeletion seems to be frequently associated with various degrees of intellectual disability, usually not a typical symptom of idiopathic generalized epilepsies (Muhle et al., 2011). However, the full phenotypic range of this microdeletion triad has not been described. Data on the frequency of these microdeletions in various pediatric epilepsies and rare epilepsy syndromes is not available. In this study we investigate the overall frequency of 15q11.2, 15q13.3 and 16p13.11 microdeletions in a heterogeneous cohort of pediatric epilepsy patients and describe the range of phenotypes associated with the three microdeletions.
We screened a diverse cohort of 570 patients with pediatric epilepsies for microdeletions at 15q11.2, 15q13.3 and 16p13.11 using quantitative polymerase chain reaction (qPCR) as previously described (Dibbens et al., 2009). For the 15q11.2 microdeletion, a probe was designed for the first intron of the CYFIP1 gene. For the 16p13.11, the probe was located in the first intron of the NDE1 gene. Data was analyzed using the SDS2.3 (Applied Biosystems) and CopyCaller 0.152 (Applied Biosystems). In brief, the delta—delta ct method was used with cut-offs of 1 for deletions and 3 for duplications. Samples were analyzed as quadruplicates and normalized to a positive control within the CopyCaller Software.
Materials and methods Patients The cohort included 101 patients with idiopathic (genetic) generalized epilepsies (Berg et al., 2010), 20 patients with idiopathic focal epilepsies, 126 patients with feverassociated epilepsy syndromes including Febrile Seizures (FS), febrile seizures plus (FS+) and genetic epilepsy with febrile seizures plus (GEFS+). 201 patients had other diverse epilepsy syndromes including Dravet syndrome, symptomatic and cryptogenic focal epilepsies and unclassified epilepsies. 122 children had a single unclassified seizures or epilepsy-associated EEG patterns without seizures. 131/570 patients (23.0%) had mild, moderate or severe intellectual disability (ID) (Table 1). Apart from few exceptions, the vast majority of patients were of Western European ancestry. The patients carrying a 15q13.3 microdeletion have been included as a case series in a previous publication (Muhle et al., 2011). The study was approved by the local ethics-committee at the University of Kiel. Written consent was obtained by the parents.
Array-CGH Identified microdeletions were confirmed using array comparative hybridization as previously described (Helbig et al., 2009).
Statistical analysis Descriptive statistics are used to demonstrate the relative frequencies of deletion carriers. Association analyses between genotype and phenotype were carried out by twosided Fisher’s exact tests. Relative risks are expressed as odds ratios plus 95% confidence intervals.
Results Phenotypes of microdeletion carriers Ten microdeletions in 15q11.2 (n = 3), 15q13.3 (n = 3) and 16p13.11 (n = 4) were identified in the overall cohort of 570 probands (1.8%). 9/10 microdeletions (15q11.2 (n = 3), 15q13.3 (n = 3) and 16p13.11 (n = 3)) were identified in patients with IGE (6/101, 6.0%) or patients with generalized EEG patterns without seizures (3/122, 2.5%) including generalized spikewaves (GSW) and photoparoxysmal response (PPR). 1/10 microdeletion was identified in the cohort of other epilepsy syndromes in a patient with West syndrome (1/201, 15q11.2 (n = 0), 15q13.3 (n = 0) and 16p13.11 (n = 1)). In the 146 patients with idiopathic focal epilepsies or fever related epilepsy syndromes (FS, FS+ or GEFS+) microdeletions were absent. The difference in frequency of microdeletions in patients with IGE patterns and other phenotypes was significant (pvalue = 2.367e−06, OR 23.8, 95% CI [6.6—79.7] Fisher’s exact test).
Overview of phenotypes and microdeletions in study cohort.
Overview of epilepsy cohort (n = 570)
Total
ID
IGE (n = 101)
MAE BMEI EOAE CAE JAE JME GTCS alone Unclassified IGE
5 1 5 19 8 9 45 10
3 1 2 2 2 0 1 5
EEG abnormalities (n = 122)
Generalized spike-wave (no seizures) Photoparoxysmal response (no seizures)
Number and type of microdeletion
Frequency
Statisticsa (other epilepsies)
Statisticsb (controls)
6
6/101; 6%
p-Value = 0.003 95% CI [1.7—35.9] OR 7.3
pValue = 2.367e−06 95% CI [6.6—79.7] OR 23.8
0 0 0 1 (15q13.3) 2 (2 × 15q13.3) 0 0 3 (2 × 15q11.2/16p13.11)
0 0 0 1/19; 5.3% 2/8; 25% 0 0 3/10; 30%
3
3/122; 2.5%
p-Value = 0.453
p-Value = 0.007 95% CI [1.6—40.2] OR 9.5
p-Value = 1.0
p-Value = 1.0
26 96
3 5
2 (15q11.2/16p13.11) 1 (16p13.11)
2/26; 7.7% 1/96; 1%
10
1
0 0
0/20; 0% 0
10
5
0
0 1/201, 0.5% 0 1/11; 9.1% 0 0 0
p-Value = 0.439
16 11 34 23 10
1 0 1 (16p13.11) 0 0 0
p-Value = 0.108
16 11 63 73 37
0/126; 0% 0 0 0
p-Value = 1.0
2 1 4
0 0 0 0
p-Value = 0.128
102 10 14
10
10/570; 1.8%
Idiopathic focal epilepsies (n = 20) BETCS/Panayiotopolous syndrome ABPE Other epilepsies (n = 201) Dravet syndrome West syndrome Symptomatic focal epilepsies Cryptogenic focal epilepsies Unclassified epilepsies Fever-associated epilepsy syndromes (n = 126) FS alone FS plus (additional afebrile seizures in patient) GEFS plus (additional afebrile seizures in relatives) Total
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This table displays the distribution of phenotypes, the frequency and type of microdeletions within the cohort and summarizes the statistical analysis. Abbreviations in order of appearance: IGE, idiopathic (genetic) generalized epilepsy; MAE, myoclonic astatic epilepsy (Doose syndrome); BMEI, benign myoclonic epilepsy of infancy; EOAE, early onset absence epilepsy (onset < 4 years); CAE, childhood absence epilepsy (onset < 10 years); JAE, juvenile absence epilepsy; JME, juvenile myoclonic epilepsy (Janz syndrome); GTCS, generalized tonic—clonic seizure; BECTS, benign epilepsy with centrotemporal spikes (rolandic epilepsy); ABPE, atypical benign partial epilepsy (Pseudo-Lennox syndrome); FS, febrile seizure; GEFS plus, genetic epilepsy with febrile seizures plus; CI, confidence interval, OR, Odds ratio. a This column demonstrates the statistical analysis of frequencies of microdeletions 15q11.2, 15q13.3 and 16p.13.11 in different epilepsy subtypes compared to frequencies in the overall group (e.g. IGE versus non-IGE). b This column summarizes the statistical analysis of frequencies of microdeletions 15q11.2, 15q13.3 and 16p.13.11 in different epilepsy subtypes compared to frequencies in controls (added for all three microdeletions: 8/3022 controls) according to (De Kovel et al., 2009). All comparisons were done by Fisher’s exact tests.
Iterative phenotyping of 15q11.2, 15q13.3 and 16p13.11 microdeletion carriers
Table 1
112 6/10 microdeletion carriers had various degrees of ID. The frequency of microdeletions in patients with epilepsy and ID was higher (6/131, 4.6%) compared to epilepsy patients with normal intellect (4/439, 0.9%, p = 0.01, OR 5.20, 95% CI [1.21—25.45] Fisher’s exact test). 6/10 microdeletion carriers had various degrees of PPR. The frequency of microdeletions in PPR was not significantly elevated (6/238 PPR positives with microdeletions vs. 4/332 without PPR, p = 0.75). A summary of the phenotypes of all patients carrying microdeletions is given in Table 2.
Segregation Segregation of variants for which parents were available for testing (6/10) is shown in Fig. 1. While 2/6 variants occurred de novo, four variants were transmitted from parents. Two variant were transmitted from unaffected parents, and two from parents with epilepsy. The phenotypes of the microdeletions carriers are detailed below. All phenotypes were challenging to classify according to the current ILAE classification scheme.
15q11.2 microdeletion (three patients) Patient #30, the daughter of unrelated healthy parents, had four generalized tonic seizures between the age of 5 and 8 years. Upon introduction of lamotrigine, she became seizure-free. EEG at seizure onset showed frontally pronounced generalized spike waves and poly-spike waves as well as PPR grade IV. Cognitive development was normal. Her paternal grandfather had seizures during childhood. The 15q11.2 variant was transmitted from the unaffected mother. Patient #54, the son of non-consanguineous parents, had typical absences and atonic seizures starting at the age of four. The EEG at seizure onset revealed irregular generalized spike-wave paroxysms. Treatment with valproic acid (VPA) and ethosuximide resulted in seizure freedom. At the age of nine, AED treatment was discontinued and the patient remained seizure free until the last consultation at the age of 19. The patient had mild intellectual disability. The 15q11.2 variant was transmitted from the mother, who had had absence epilepsy treated with VPA during pregnancy. Patient #326, the daughter of unrelated healthy parents, had irregular generalized spike waves in EEG which was performed for a diagnostic work up of headaches. She never experienced epileptic seizures and had mild intellectual disability. The 15q11.2 variant was transmitted from the unaffected father. The patient’s older sister, who also carried the variant, had idiopathic generalized epilepsy with rare myoclonic seizures and GTCS. The sister’s EEG showed irregular generalized spike waves and PPR grade IV. Also, the paternal grandfather had epilepsy, but was not available for genetic testing, detailed phenotyping was not possible.
15q13.3 microdeletion (three patients) The three patients with 15q13.3 microdeletions were previously described (Muhle et al., 2011), but were included
J.A. Jähn et al. in the current study to assess the overall frequency of these variants. In brief, patient #10, male, presented with absence seizures at the age of seven. During adolescence one GTCS occurred. EEG showed 3-Hz-spike wave paroxysms. At the age of eight, moderate ID was noted. Patient #237, male, had absence seizures since the age of 13 and 3-Hz-spike wave paroxysms as well as PPR grade IV in EEG. He had mild intellectual disability. Patient #490, female, had one GTCS followed by absence status at the age of 17 with continuous 2.5- to 4-Hz-spike waves in EEG. Mild intellectual disability was noted in early childhood. Pedigrees of the three patients with 15q13.3 deletions are shown in Fig. 1.
16p13.11 microdeletion (four patients) Patient #51 is the son of non-consanguineous healthy parents. He started to have tonic seizures at the age of two days due to bacterial meningitis. Seizures during neonatal period were controlled by phenobarbital. At the age of seven months, complex partial seizures were noted. EEG revealed multifocal sharp waves, MRI showed a lesion in the left temporal lobe. At the age of two years, seizures with head nodding and tonic posturing of the arms were observed, the EEG showed hypsarrhythmia. Seizures and EEG were resistant to treatment with multiple AEDs, but were eventually controlled on dexamethasone at the age of four years. The patient has been seizure free until the last consultation at the age of nine. A cognitive decline was noted at the age of two years, early development was normal. At the last consultation the patient had mild intellectual disability. Parents were not available for testing. Patient #141 is the son of non-consanguineous parents. His EEG at the age of 12 years showed generalized spike waves and PPR grade IV. The EEG was performed for diagnostic workup of episodes suspicious of myoclonic seizures. No seizures were observed during several EEG-recordings including long-term EEG. The patient was of normal intelligence. The 16p13.11 variant was found to be inherited from the mother, who had GTCS. The father was not available for testing. Patient #349 is the daughter of non-consanguineous healthy parents. She had PPR grade II on EEG, but no epileptic seizures. Her intelligence was normal. Patient #505 is the daughter of healthy parents. She had photic-induced GTCS and absence seizures with eyelid myoclonia starting at the age of 14. Multiple EEGs recorded between the age of 14 and 18 revealed irregular generalized spike wave paroxysms and PPR grade IV. The patient was of normal intelligence. Her paternal uncle had photic induced GTCS.
Discussion In this study, we investigated the frequency of 15q11.2, 15q13.3 and 16p13.11 microdeletions in a heterogeneous cohort of pediatric epilepsy patients and described the phenotypes associated with the three microdeletions. In accordance with previous studies (Heinzen et al., 2010; De Kovel et al., 2009), the overall frequency of 15q11.2, 15q13.3 and 16p13.11 microdeletions in our epilepsy cohort
Phenotype of patients carrying a microdeletion.
Patient
Gender
Microdeletion
Epilepsy syndrome
Age at epilepsy onset
Seizure types
EEG
Intellectual status
Inheritance (parental epilepsy syndrome)
#30
f
15q11.2
IGE, unclassified
5 years
Maternal (unaffected)
m
15q11.2
IGE, unclassified
4 years
Generalized sw, PPR IV Generalized sw
Normal
#54
Mild ID
#326 #10
f m
15q11.2 15q13.3, BP4-BP5
EEG discharges only CAE
— 7 years
Generalized tonic sz Absences, atonic sz — Absences, GTCS
Generalized sw 3 Hz. sw
#237 #490
m f
15q13.3, BP4-BP5 15q13.3, BP3-BP5
JAE JAE
13 years 17 years
Normal Moderate ID Mild ID Mild ID
Maternal (absence-epilepsy) Paternal (unaffected) Paternal (juvenile sz., not specified) Unknown Sporadic
#51
m
16p13.11
West syndrome
2 days
Mild ID
Unknown
#141
m
16p13.11
EEG discharges only
—
—
Multifocal sharp waves, hypsarrhythmia Generalized sw
Normal
#349 #505
f f
16p13.11 16p13.11
EEG discharges only IGE, unclassified
— 14 years
— Photic-induced GTCS, absences with eyelid myoclonia
PPR II Generalized sw, PPR IV
Normal Normal
Maternal (IGE with GTCS only) Unknown Unknown
Absences GTCS, absence status Tonic sz, CPS, infantile spams
3 Hz. sw, PPR IV 2.5- to 4-Hz. sw
Iterative phenotyping of 15q11.2, 15q13.3 and 16p13.11 microdeletion carriers
Table 2
Phenotypic description of microdeletion carriers. To further specify the deletions, breakpoints are listed for the 15q13.3 deletion. Abbreviations in order of appearance: f, female; m, male; BP, breakpoint, IGE, idiopathic generalized epilepsy; EEG, electroencephalogram; CAE, childhood absence epilepsy; JAE, juvenile absence epilepsy; sz, seizures; GTCS, generalized tonic—clonic seizures; CPS, complex partial (dyscognitive) seizures; sw, spike-waves; PPR, photoparoxysmal response; Hz, Hertz; ID, intellectual disability.
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Figure 1 Segregation of CNVs: availability of parents in 6/10 patients. 2/6 variants occurred de novo, 2 variants were transmitted from unaffected parents, 2 from parents with epilepsy. The affected sister of patient #326 also carried the 15q11.2 variant while the brother of patient #10, who had intellectual disability (ID), but no epilepsy or generalized EEG abnormalities and PPR, did not have a 15q13.3 microdeletion.
was 1.8%. In contrast, these copy number variants have been detected at significantly lower frequencies in control populations (De Kovel et al., 2009). Iterative phenotyping revealed a wide range of generalized epilepsy phenotypes in microdeletion carriers. All phenotypes were challenging to classify according to the current ILAE classification scheme due to unusual phenotypic features. For the 15q13.3 microdeletion, this is discussed in detail in (Muhle et al., 2011). In our pediatric cohort, we observed several phenomena that have previously not been reported in the literature. First, 16p13.11 microdeletions in our cohort were only found in patients with IGE or generalized EEG patterns and in one patient with West syndrome, but not in patients with focal epilepsies. This is different to the findings of Heinzen et al. (Heinzen et al., 2010), who described deletions in partial and generalized epilepsies. As it is known that PPR may disappear spontaneously during childhood and adolescence (Jeavons et al., 1986), 16p13.11 microdeletion carriers in the previous study might have had PPR or generalized discharges during childhood. Secondly, in our cohort the microdeletion rate in patients with epilepsy and ID was significantly higher than in patients with epilepsy and normal intellect. Similarly, Hannes et al. (2009) reported 16p13.11 microdeletions in patients with ID and epilepsy, whereas others did not report ID as a common feature in epilepsy patients carrying microdeletions at 16p13.11 (Heinzen et al., 2010). We suggest that the precise frequency
of 16p13.11 microdeletion carriers with ID may depend on ascertainment of the study cohort, i.e. the overall frequency of more severe 16p13.11 phenotypes that are associated with microdeletions at 16p13.11 might be higher in pediatric cohorts. Third, microdeletions at the three investigated loci were absent in patients with idiopathic focal epilepsies and in patients with fever-related epilepsy phenotypes, even though some of the subgroups were relatively small and not sufficiently powered to detect variants at a low frequency. Particularly in fever-related epilepsy phenotypes, the frequency of the microdeletion triad has not been investigated before. With regards to focal epilepsies, previous studies have described several patients with focal epilepsies and microdeletions at 15q11.2 and 16p13.11 (Heinzen et al., 2010). However, none of these patients were classified as idiopathic focal epilepsies. Segregation of variants showed a mixed picture that has previously been reported for all three variants including de novo deletions as well as transmission from affected and unaffected parents (De Kovel et al., 2010). This observation is in line with the segregation expected from rare genetic variants (Helbig et al., 2009) and is explained by the interpretation of these variants as risk factors rather than causal factors. Of note, both de novo events are deletions at 15q13.3 and this microdeletion represents the strongest genetic risk factor amongst the variants investigated.
Iterative phenotyping of 15q11.2, 15q13.3 and 16p13.11 microdeletion carriers Previous studies have discussed in detail possible pathomechanisms of these microdeletions leading to various epilepsy syndromes and a variety of other neuropsychiatric phenotypes (De Kovel et al., 2009; Heinzen et al., 2010; Helbig et al., 2009). Haploinsufficiency is assumed to be the main mechanism by which a 50% reduction of the candidate gene (CYFIP1 and others for microdeletion 15q11.2, CHRNA7 and others for 15q13.3, NDE1 and others for 16p13.11) causes significant reduction in the amount of the respective proteins (De Kovel et al., 2009; Heinzen et al., 2010). Additional genetic variation might explain the phenotypic variability of all three microdeletions as suggested by a significantly higher burden of large heterozygous deletions in epilepsy patients compared to controls (Heinzen et al., 2010). Within this study, we concentrated on known, recurrent microdeletions. Therefore, we cannot comment on the overall CNV load including rare copy number variations in the cohort. The frequency of additional, rare CNVs might be higher as indicated by previous studies (Mefford et al., 2010, 2011).
Conclusions In conclusion, in our pediatric cohort, recurrent microdeletions at 15q11.2, 15q13.3 and 16p13.11 are mainly associated with phenotypes of the spectrum of idiopathic generalized epilepsies or related generalized EEG patterns. In contrast to previous reports, these recurrent microdeletions are virtually absent in focal epilepsies, FS, FS+ and GEFS+. Microdeletion carriers have a five-fold risk to present with various degrees of ID compared to patients without these risk factors. This microdeletion triad might help delineate a novel spectrum of epilepsy phenotypes classifiable through clinical, electrographic and genetic data.
Acknowledgements The authors would like to thank the patients and their parents for participation in this study. We are grateful to Angelika Ackerhans and Klaus Moldenhauer for database management and Anita Dietsch, Catharina Fürstenau, Katja Schmidt-Cloppenborg, Ilona Urbach and Tanja Wesse for technical assistance.
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H., De Jonghe, P., Suls, A., Ruckert, I.M., Wichmann, H.E., Franke, A., Schreiber, S., Nurnberg, P., Elger, C.E., Lerche, H., Stephani, U., Koeleman, B.P., Lindhout, D., Eichler, E.E., Sander, T., 2009. Recurrent microdeletions at 15q11.2 and 16p13.11 predispose to idiopathic generalized epilepsies. Brain 133, 23—32. De Kovel, C.G., Trucks, H., Helbig, I., Mefford, H.C., Baker, C., Leu, C., Kluck, C., Muhle, H., Von Spiczak, S., Ostertag, P., Obermeier, T., Kleefuss-Lie, A.A., Hallmann, K., Steffens, M., Gaus, V., Klein, K.M., Hamer, H.M., Rosenow, F., Brilstra, E.H., Trenite, D.K., Swinkels, M.E., Weber, Y.G., Unterberger, I., Zimprich, F., Urak, L., Feucht, M., Fuchs, K., Moller, R.S., Hjalgrim, H., De Jonghe, P., Suls, A., Ruckert, I.M., Wichmann, H.E., Franke, A., Schreiber, S., Nurnberg, P., Elger, C.E., Lerche, H., Stephani, U., Koeleman, B.P., Lindhout, D., Eichler, E.E., Sander, T., 2010. Recurrent microdeletions at 15q11.2 and 16p13.11 predispose to idiopathic generalized epilepsies. Brain 133, 23—32. Dibbens, L.M., Mullen, S., Helbig, I., Mefford, H.C., Bayly, M.A., Bellows, S., Leu, C., Trucks, H., Obermeier, T., Wittig, M., Franke, A., Caglayan, H., Yapici, Z., Sander, T., Eichler, E.E., Scheffer, I.E., Mulley, J.C., Berkovic, S.F., 2009. Familial and sporadic 15q13.3 microdeletions in idiopathic generalized epilepsy: precedent for disorders with complex inheritance. Hum. Mol. Genet. 18, 3626—3631. Hannes, F.D., Sharp, A.J., Mefford, H.C., De Ravel, T., Ruivenkamp, C.A., Breuning, M.H., Fryns, J.P., Devriendt, K., Van Buggenhout, G., Vogels, A., Stewart, H., Hennekam, R.C., Cooper, G.M., Regan, R., Knight, S.J., Eichler, E.E., Vermeesch, J.R., 2009. Recurrent reciprocal deletions and duplications of 16p13.11: the deletion is a risk factor for MR/MCA while the duplication may be a rare benign variant. J. Med. Genet. 46, 223—232. Heinzen, E.L., Radtke, R.A., Urban, T.J., Cavalleri, G.L., Depondt, C., Need, A.C., Walley, N.M., Nicoletti, P., Ge, D., Catarino, C.B., Duncan, J.S., Kasperaviciute, D., Tate, S.K., Caboclo, L.O., Sander, J.W., Clayton, L., Linney, K.N., Shianna, K.V., Gumbs, C.E., Smith, J., Cronin, K.D., Maia, J.M., Doherty, C.P., Pandolfo, M., Leppert, D., Middleton, L.T., Gibson, R.A., Johnson, M.R., Matthews, P.M., Hosford, D., Kalviainen, R., Eriksson, K., Kantanen, A.M., Dorn, T., Hansen, J., Kramer, G., Steinhoff, B.J., Wieser, H.G., Zumsteg, D., Ortega, M., Wood, N.W., HuxleyJones, J., Mikati, M., Gallentine, W.B., Husain, A.M., Buckley, P.G., Stallings, R.L., Podgoreanu, M.V., Delanty, N., Sisodiya, S.M., Goldstein, D.B., 2010. Rare deletions at 16p13.11 predispose to a diverse spectrum of sporadic epilepsy syndromes. Am. J. Hum. Genet. 86, 707—718. Helbig, I., Mefford, H.C., Sharp, A.J., Guipponi, M., Fichera, M., Franke, A., Muhle, H., De Kovel, C., Baker, C., Von Spiczak, S., Kron, K.L., Steinich, I., Kleefuss-Lie, A.A., Leu, C., Gaus, V., Schmitz, B., Klein, K.M., Reif, P.S., Rosenow, F., Weber, Y., Lerche, H., Zimprich, F., Urak, L., Fuchs, K., Feucht, M., Genton, P., Thomas, P., Visscher, F., De Haan, G.J., Moller, R.S., Hjalgrim, H., Luciano, D., Wittig, M., Nothnagel, M., Elger, C.E., Nurnberg, P., Romano, C., Malafosse, A., Koeleman, B.P., Lindhout, D., Stephani, U., Schreiber, S., Eichler, E.E., Sander, T., 2009. 15q13.3 microdeletions increase risk of idiopathic generalized epilepsy. Nat. Genet. 41, 160—162. Jeavons, P.M., Bishop, A., Harding, G.F., 1986. The prognosis of photosensitivity. Epilepsia 27, 569—575. Mefford, H.C., Muhle, H., Ostertag, P., Von Spiczak, S., Buysse, K., Baker, C., Franke, A., Malafosse, A., Genton, P., Thomas, P., Gurnett, C.A., Schreiber, S., Bassuk, A.G., Guipponi, M., Stephani, U., Helbig, I., Eichler, E.E., 2010. Genome-wide copy number variation in epilepsy: novel susceptibility loci in idiopathic generalized and focal epilepsies. PLoS Genet. 6, e1000962. Mefford, H.C., Yendle, S.C., Hsu, C., Cook, J., Geraghty, E., Mcmahon, J.M., Eeg-Olofsson, O., Sadleir, L.G., Gill, D., Ben-Zeev, B., Lerman-Sagie, T., Mackay, M., Freeman,
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J.A. Jähn et al. Muhle, H., Mefford, H.C., Obermeier, T., Von Spiczak, S., Eichler, E.E., Stephani, U., Sander, T., Helbig, I., 2011. Absence seizures with intellectual disability as a phenotype of the 15q13.3 microdeletion syndrome. Epilepsia 52, e194—e198.
journal homepage: www.elsevier.com/locate/epilepsyres
Iterative phenotyping of 15q11.2, 15q13.3 and 16p13.11 microdeletion carriers in pediatric epilepsies Johanna A. Jähn a,1, Sarah von Spiczak a,b,1, Hiltrud Muhle a, Tanja Obermeier a, Andre Franke c, Heather C. Mefford d, Ulrich Stephani a,b, Ingo Helbig a,∗ a
Department of Neuropediatrics, University Medical Center Schleswig-Holstein (UKSH, Campus Kiel), Kiel, Germany b Northern German Epilepsy Center for Children and Adolescents, Schwentinental-Raisdorf, Germany c Institute of Clinical Molecular Biology, Christian-Albrechts-University Kiel, Kiel, Germany d Department of Pediatrics, Division of Genetic Medicine, University of Washington, Seattle, WA, USA Received 18 February 2013 ; received in revised form 16 September 2013; accepted 13 October 2013 Available online 26 October 2013
KEYWORDS Copy number variation; 15q11.2; 15q13.3; 16p13.11 microdeletions
Summary Microdeletions at 15q11.2, 15q13.3 and 16p13.11 are known genetic risk factors for idiopathic generalized epilepsies and other neurodevelopmental disorders. The full phenotypic range of this microdeletion triad in pediatric epilepsies is unknown. We attempted to describe associated phenotypes in a cohort of pediatric epilepsy patients. We screened 570 patients with pediatric epilepsies including idiopathic generalized epilepsies, focal epilepsies and fever-associated epilepsy syndromes for microdeletions at 15q11.2, 15q13.3 and 16p13.11 using quantitative polymerase chain reaction. Identified microdeletions were confirmed using array comparative hybridization. Ten microdeletions in 15q11.2 (n = 3), 15q13.3 (n = 3) and 16p13.11 (n = 4) were identified (1.8%). 9/10 microdeletions were identified in patients with IGE (6/101, 6.0%) or patients with generalized EEG patterns without seizures (3/122, 2.5%). 6/10 microdeletion carriers had various degrees of ID; the frequency of microdeletions in patients with epilepsy and ID was higher (4.6%) compared to patients with normal intellect (0.9%). Iterative phenotyping revealed a wide range of generalized epilepsy phenotypes. In our pediatric cohort, recurrent microdeletions at 15q11.2, 15q13.3 and 16p13.11 are mainly associated with phenotypes related to idiopathic generalized epilepsies or related EEG patterns. In contrast to
∗ Corresponding author at: Department of Neuropediatrics, University Medical Center Schleswig-Holstein (UKSH, Campus Kiel), ArnoldHeller-Str. 3, Haus 9, D-24105 Kiel, Germany. Tel.: +49 431 597 1761; fax: +49 431 597 1769. E-mail addresses: [email protected] (J.A. Jähn), [email protected] (S. von Spiczak), [email protected] (H. Muhle), [email protected] (T. Obermeier), [email protected] (A. Franke), [email protected] (H.C. Mefford), [email protected] (I. Helbig). 1 These authors have contributed equally.
0920-1211/$ — see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.eplepsyres.2013.10.001
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J.A. Jähn et al. previous reports, these recurrent microdeletions are virtually absent in focal epilepsies, FS, FS+ and GEFS+. Microdeletion carriers have a five-fold risk to present with various degrees of ID compared to patients without these risk factors. This microdeletion triad might help delineate a novel spectrum of epilepsy phenotypes classifiable through clinical, electrographic and genetic data. © 2013 Elsevier B.V. All rights reserved.
Introduction
qPCR
Microdeletions and microduplications are known genetic risk factors in common diseases including neuropsychiatric disorders such as schizophrenia, autism or intellectual disability. Rare and recurrent copy number variations (CNV) were also found to be associated with various epilepsy phenotypes. Microdeletions at 15q11.2, 15q13.3 and 16p13.11 are the most common CNVs in epilepsies occurring with a frequency of about 1,5% (Helbig et al., 2009; De Kovel et al., 2010). While microdeletions at 15q13.3 have only been described in association with idiopathic (genetic) generalized epilepsies (De Kovel et al., 2010), microdeletions at 15q11.2 and 16p13.11 were also found in generalized and focal epilepsy phenotypes (Heinzen et al., 2010). Notably, absence epilepsy in patients carrying a 15q13.3 microdeletion seems to be frequently associated with various degrees of intellectual disability, usually not a typical symptom of idiopathic generalized epilepsies (Muhle et al., 2011). However, the full phenotypic range of this microdeletion triad has not been described. Data on the frequency of these microdeletions in various pediatric epilepsies and rare epilepsy syndromes is not available. In this study we investigate the overall frequency of 15q11.2, 15q13.3 and 16p13.11 microdeletions in a heterogeneous cohort of pediatric epilepsy patients and describe the range of phenotypes associated with the three microdeletions.
We screened a diverse cohort of 570 patients with pediatric epilepsies for microdeletions at 15q11.2, 15q13.3 and 16p13.11 using quantitative polymerase chain reaction (qPCR) as previously described (Dibbens et al., 2009). For the 15q11.2 microdeletion, a probe was designed for the first intron of the CYFIP1 gene. For the 16p13.11, the probe was located in the first intron of the NDE1 gene. Data was analyzed using the SDS2.3 (Applied Biosystems) and CopyCaller 0.152 (Applied Biosystems). In brief, the delta—delta ct method was used with cut-offs of 1 for deletions and 3 for duplications. Samples were analyzed as quadruplicates and normalized to a positive control within the CopyCaller Software.
Materials and methods Patients The cohort included 101 patients with idiopathic (genetic) generalized epilepsies (Berg et al., 2010), 20 patients with idiopathic focal epilepsies, 126 patients with feverassociated epilepsy syndromes including Febrile Seizures (FS), febrile seizures plus (FS+) and genetic epilepsy with febrile seizures plus (GEFS+). 201 patients had other diverse epilepsy syndromes including Dravet syndrome, symptomatic and cryptogenic focal epilepsies and unclassified epilepsies. 122 children had a single unclassified seizures or epilepsy-associated EEG patterns without seizures. 131/570 patients (23.0%) had mild, moderate or severe intellectual disability (ID) (Table 1). Apart from few exceptions, the vast majority of patients were of Western European ancestry. The patients carrying a 15q13.3 microdeletion have been included as a case series in a previous publication (Muhle et al., 2011). The study was approved by the local ethics-committee at the University of Kiel. Written consent was obtained by the parents.
Array-CGH Identified microdeletions were confirmed using array comparative hybridization as previously described (Helbig et al., 2009).
Statistical analysis Descriptive statistics are used to demonstrate the relative frequencies of deletion carriers. Association analyses between genotype and phenotype were carried out by twosided Fisher’s exact tests. Relative risks are expressed as odds ratios plus 95% confidence intervals.
Results Phenotypes of microdeletion carriers Ten microdeletions in 15q11.2 (n = 3), 15q13.3 (n = 3) and 16p13.11 (n = 4) were identified in the overall cohort of 570 probands (1.8%). 9/10 microdeletions (15q11.2 (n = 3), 15q13.3 (n = 3) and 16p13.11 (n = 3)) were identified in patients with IGE (6/101, 6.0%) or patients with generalized EEG patterns without seizures (3/122, 2.5%) including generalized spikewaves (GSW) and photoparoxysmal response (PPR). 1/10 microdeletion was identified in the cohort of other epilepsy syndromes in a patient with West syndrome (1/201, 15q11.2 (n = 0), 15q13.3 (n = 0) and 16p13.11 (n = 1)). In the 146 patients with idiopathic focal epilepsies or fever related epilepsy syndromes (FS, FS+ or GEFS+) microdeletions were absent. The difference in frequency of microdeletions in patients with IGE patterns and other phenotypes was significant (pvalue = 2.367e−06, OR 23.8, 95% CI [6.6—79.7] Fisher’s exact test).
Overview of phenotypes and microdeletions in study cohort.
Overview of epilepsy cohort (n = 570)
Total
ID
IGE (n = 101)
MAE BMEI EOAE CAE JAE JME GTCS alone Unclassified IGE
5 1 5 19 8 9 45 10
3 1 2 2 2 0 1 5
EEG abnormalities (n = 122)
Generalized spike-wave (no seizures) Photoparoxysmal response (no seizures)
Number and type of microdeletion
Frequency
Statisticsa (other epilepsies)
Statisticsb (controls)
6
6/101; 6%
p-Value = 0.003 95% CI [1.7—35.9] OR 7.3
pValue = 2.367e−06 95% CI [6.6—79.7] OR 23.8
0 0 0 1 (15q13.3) 2 (2 × 15q13.3) 0 0 3 (2 × 15q11.2/16p13.11)
0 0 0 1/19; 5.3% 2/8; 25% 0 0 3/10; 30%
3
3/122; 2.5%
p-Value = 0.453
p-Value = 0.007 95% CI [1.6—40.2] OR 9.5
p-Value = 1.0
p-Value = 1.0
26 96
3 5
2 (15q11.2/16p13.11) 1 (16p13.11)
2/26; 7.7% 1/96; 1%
10
1
0 0
0/20; 0% 0
10
5
0
0 1/201, 0.5% 0 1/11; 9.1% 0 0 0
p-Value = 0.439
16 11 34 23 10
1 0 1 (16p13.11) 0 0 0
p-Value = 0.108
16 11 63 73 37
0/126; 0% 0 0 0
p-Value = 1.0
2 1 4
0 0 0 0
p-Value = 0.128
102 10 14
10
10/570; 1.8%
Idiopathic focal epilepsies (n = 20) BETCS/Panayiotopolous syndrome ABPE Other epilepsies (n = 201) Dravet syndrome West syndrome Symptomatic focal epilepsies Cryptogenic focal epilepsies Unclassified epilepsies Fever-associated epilepsy syndromes (n = 126) FS alone FS plus (additional afebrile seizures in patient) GEFS plus (additional afebrile seizures in relatives) Total
111
This table displays the distribution of phenotypes, the frequency and type of microdeletions within the cohort and summarizes the statistical analysis. Abbreviations in order of appearance: IGE, idiopathic (genetic) generalized epilepsy; MAE, myoclonic astatic epilepsy (Doose syndrome); BMEI, benign myoclonic epilepsy of infancy; EOAE, early onset absence epilepsy (onset < 4 years); CAE, childhood absence epilepsy (onset < 10 years); JAE, juvenile absence epilepsy; JME, juvenile myoclonic epilepsy (Janz syndrome); GTCS, generalized tonic—clonic seizure; BECTS, benign epilepsy with centrotemporal spikes (rolandic epilepsy); ABPE, atypical benign partial epilepsy (Pseudo-Lennox syndrome); FS, febrile seizure; GEFS plus, genetic epilepsy with febrile seizures plus; CI, confidence interval, OR, Odds ratio. a This column demonstrates the statistical analysis of frequencies of microdeletions 15q11.2, 15q13.3 and 16p.13.11 in different epilepsy subtypes compared to frequencies in the overall group (e.g. IGE versus non-IGE). b This column summarizes the statistical analysis of frequencies of microdeletions 15q11.2, 15q13.3 and 16p.13.11 in different epilepsy subtypes compared to frequencies in controls (added for all three microdeletions: 8/3022 controls) according to (De Kovel et al., 2009). All comparisons were done by Fisher’s exact tests.
Iterative phenotyping of 15q11.2, 15q13.3 and 16p13.11 microdeletion carriers
Table 1
112 6/10 microdeletion carriers had various degrees of ID. The frequency of microdeletions in patients with epilepsy and ID was higher (6/131, 4.6%) compared to epilepsy patients with normal intellect (4/439, 0.9%, p = 0.01, OR 5.20, 95% CI [1.21—25.45] Fisher’s exact test). 6/10 microdeletion carriers had various degrees of PPR. The frequency of microdeletions in PPR was not significantly elevated (6/238 PPR positives with microdeletions vs. 4/332 without PPR, p = 0.75). A summary of the phenotypes of all patients carrying microdeletions is given in Table 2.
Segregation Segregation of variants for which parents were available for testing (6/10) is shown in Fig. 1. While 2/6 variants occurred de novo, four variants were transmitted from parents. Two variant were transmitted from unaffected parents, and two from parents with epilepsy. The phenotypes of the microdeletions carriers are detailed below. All phenotypes were challenging to classify according to the current ILAE classification scheme.
15q11.2 microdeletion (three patients) Patient #30, the daughter of unrelated healthy parents, had four generalized tonic seizures between the age of 5 and 8 years. Upon introduction of lamotrigine, she became seizure-free. EEG at seizure onset showed frontally pronounced generalized spike waves and poly-spike waves as well as PPR grade IV. Cognitive development was normal. Her paternal grandfather had seizures during childhood. The 15q11.2 variant was transmitted from the unaffected mother. Patient #54, the son of non-consanguineous parents, had typical absences and atonic seizures starting at the age of four. The EEG at seizure onset revealed irregular generalized spike-wave paroxysms. Treatment with valproic acid (VPA) and ethosuximide resulted in seizure freedom. At the age of nine, AED treatment was discontinued and the patient remained seizure free until the last consultation at the age of 19. The patient had mild intellectual disability. The 15q11.2 variant was transmitted from the mother, who had had absence epilepsy treated with VPA during pregnancy. Patient #326, the daughter of unrelated healthy parents, had irregular generalized spike waves in EEG which was performed for a diagnostic work up of headaches. She never experienced epileptic seizures and had mild intellectual disability. The 15q11.2 variant was transmitted from the unaffected father. The patient’s older sister, who also carried the variant, had idiopathic generalized epilepsy with rare myoclonic seizures and GTCS. The sister’s EEG showed irregular generalized spike waves and PPR grade IV. Also, the paternal grandfather had epilepsy, but was not available for genetic testing, detailed phenotyping was not possible.
15q13.3 microdeletion (three patients) The three patients with 15q13.3 microdeletions were previously described (Muhle et al., 2011), but were included
J.A. Jähn et al. in the current study to assess the overall frequency of these variants. In brief, patient #10, male, presented with absence seizures at the age of seven. During adolescence one GTCS occurred. EEG showed 3-Hz-spike wave paroxysms. At the age of eight, moderate ID was noted. Patient #237, male, had absence seizures since the age of 13 and 3-Hz-spike wave paroxysms as well as PPR grade IV in EEG. He had mild intellectual disability. Patient #490, female, had one GTCS followed by absence status at the age of 17 with continuous 2.5- to 4-Hz-spike waves in EEG. Mild intellectual disability was noted in early childhood. Pedigrees of the three patients with 15q13.3 deletions are shown in Fig. 1.
16p13.11 microdeletion (four patients) Patient #51 is the son of non-consanguineous healthy parents. He started to have tonic seizures at the age of two days due to bacterial meningitis. Seizures during neonatal period were controlled by phenobarbital. At the age of seven months, complex partial seizures were noted. EEG revealed multifocal sharp waves, MRI showed a lesion in the left temporal lobe. At the age of two years, seizures with head nodding and tonic posturing of the arms were observed, the EEG showed hypsarrhythmia. Seizures and EEG were resistant to treatment with multiple AEDs, but were eventually controlled on dexamethasone at the age of four years. The patient has been seizure free until the last consultation at the age of nine. A cognitive decline was noted at the age of two years, early development was normal. At the last consultation the patient had mild intellectual disability. Parents were not available for testing. Patient #141 is the son of non-consanguineous parents. His EEG at the age of 12 years showed generalized spike waves and PPR grade IV. The EEG was performed for diagnostic workup of episodes suspicious of myoclonic seizures. No seizures were observed during several EEG-recordings including long-term EEG. The patient was of normal intelligence. The 16p13.11 variant was found to be inherited from the mother, who had GTCS. The father was not available for testing. Patient #349 is the daughter of non-consanguineous healthy parents. She had PPR grade II on EEG, but no epileptic seizures. Her intelligence was normal. Patient #505 is the daughter of healthy parents. She had photic-induced GTCS and absence seizures with eyelid myoclonia starting at the age of 14. Multiple EEGs recorded between the age of 14 and 18 revealed irregular generalized spike wave paroxysms and PPR grade IV. The patient was of normal intelligence. Her paternal uncle had photic induced GTCS.
Discussion In this study, we investigated the frequency of 15q11.2, 15q13.3 and 16p13.11 microdeletions in a heterogeneous cohort of pediatric epilepsy patients and described the phenotypes associated with the three microdeletions. In accordance with previous studies (Heinzen et al., 2010; De Kovel et al., 2009), the overall frequency of 15q11.2, 15q13.3 and 16p13.11 microdeletions in our epilepsy cohort
Phenotype of patients carrying a microdeletion.
Patient
Gender
Microdeletion
Epilepsy syndrome
Age at epilepsy onset
Seizure types
EEG
Intellectual status
Inheritance (parental epilepsy syndrome)
#30
f
15q11.2
IGE, unclassified
5 years
Maternal (unaffected)
m
15q11.2
IGE, unclassified
4 years
Generalized sw, PPR IV Generalized sw
Normal
#54
Mild ID
#326 #10
f m
15q11.2 15q13.3, BP4-BP5
EEG discharges only CAE
— 7 years
Generalized tonic sz Absences, atonic sz — Absences, GTCS
Generalized sw 3 Hz. sw
#237 #490
m f
15q13.3, BP4-BP5 15q13.3, BP3-BP5
JAE JAE
13 years 17 years
Normal Moderate ID Mild ID Mild ID
Maternal (absence-epilepsy) Paternal (unaffected) Paternal (juvenile sz., not specified) Unknown Sporadic
#51
m
16p13.11
West syndrome
2 days
Mild ID
Unknown
#141
m
16p13.11
EEG discharges only
—
—
Multifocal sharp waves, hypsarrhythmia Generalized sw
Normal
#349 #505
f f
16p13.11 16p13.11
EEG discharges only IGE, unclassified
— 14 years
— Photic-induced GTCS, absences with eyelid myoclonia
PPR II Generalized sw, PPR IV
Normal Normal
Maternal (IGE with GTCS only) Unknown Unknown
Absences GTCS, absence status Tonic sz, CPS, infantile spams
3 Hz. sw, PPR IV 2.5- to 4-Hz. sw
Iterative phenotyping of 15q11.2, 15q13.3 and 16p13.11 microdeletion carriers
Table 2
Phenotypic description of microdeletion carriers. To further specify the deletions, breakpoints are listed for the 15q13.3 deletion. Abbreviations in order of appearance: f, female; m, male; BP, breakpoint, IGE, idiopathic generalized epilepsy; EEG, electroencephalogram; CAE, childhood absence epilepsy; JAE, juvenile absence epilepsy; sz, seizures; GTCS, generalized tonic—clonic seizures; CPS, complex partial (dyscognitive) seizures; sw, spike-waves; PPR, photoparoxysmal response; Hz, Hertz; ID, intellectual disability.
113
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J.A. Jähn et al.
Figure 1 Segregation of CNVs: availability of parents in 6/10 patients. 2/6 variants occurred de novo, 2 variants were transmitted from unaffected parents, 2 from parents with epilepsy. The affected sister of patient #326 also carried the 15q11.2 variant while the brother of patient #10, who had intellectual disability (ID), but no epilepsy or generalized EEG abnormalities and PPR, did not have a 15q13.3 microdeletion.
was 1.8%. In contrast, these copy number variants have been detected at significantly lower frequencies in control populations (De Kovel et al., 2009). Iterative phenotyping revealed a wide range of generalized epilepsy phenotypes in microdeletion carriers. All phenotypes were challenging to classify according to the current ILAE classification scheme due to unusual phenotypic features. For the 15q13.3 microdeletion, this is discussed in detail in (Muhle et al., 2011). In our pediatric cohort, we observed several phenomena that have previously not been reported in the literature. First, 16p13.11 microdeletions in our cohort were only found in patients with IGE or generalized EEG patterns and in one patient with West syndrome, but not in patients with focal epilepsies. This is different to the findings of Heinzen et al. (Heinzen et al., 2010), who described deletions in partial and generalized epilepsies. As it is known that PPR may disappear spontaneously during childhood and adolescence (Jeavons et al., 1986), 16p13.11 microdeletion carriers in the previous study might have had PPR or generalized discharges during childhood. Secondly, in our cohort the microdeletion rate in patients with epilepsy and ID was significantly higher than in patients with epilepsy and normal intellect. Similarly, Hannes et al. (2009) reported 16p13.11 microdeletions in patients with ID and epilepsy, whereas others did not report ID as a common feature in epilepsy patients carrying microdeletions at 16p13.11 (Heinzen et al., 2010). We suggest that the precise frequency
of 16p13.11 microdeletion carriers with ID may depend on ascertainment of the study cohort, i.e. the overall frequency of more severe 16p13.11 phenotypes that are associated with microdeletions at 16p13.11 might be higher in pediatric cohorts. Third, microdeletions at the three investigated loci were absent in patients with idiopathic focal epilepsies and in patients with fever-related epilepsy phenotypes, even though some of the subgroups were relatively small and not sufficiently powered to detect variants at a low frequency. Particularly in fever-related epilepsy phenotypes, the frequency of the microdeletion triad has not been investigated before. With regards to focal epilepsies, previous studies have described several patients with focal epilepsies and microdeletions at 15q11.2 and 16p13.11 (Heinzen et al., 2010). However, none of these patients were classified as idiopathic focal epilepsies. Segregation of variants showed a mixed picture that has previously been reported for all three variants including de novo deletions as well as transmission from affected and unaffected parents (De Kovel et al., 2010). This observation is in line with the segregation expected from rare genetic variants (Helbig et al., 2009) and is explained by the interpretation of these variants as risk factors rather than causal factors. Of note, both de novo events are deletions at 15q13.3 and this microdeletion represents the strongest genetic risk factor amongst the variants investigated.
Iterative phenotyping of 15q11.2, 15q13.3 and 16p13.11 microdeletion carriers Previous studies have discussed in detail possible pathomechanisms of these microdeletions leading to various epilepsy syndromes and a variety of other neuropsychiatric phenotypes (De Kovel et al., 2009; Heinzen et al., 2010; Helbig et al., 2009). Haploinsufficiency is assumed to be the main mechanism by which a 50% reduction of the candidate gene (CYFIP1 and others for microdeletion 15q11.2, CHRNA7 and others for 15q13.3, NDE1 and others for 16p13.11) causes significant reduction in the amount of the respective proteins (De Kovel et al., 2009; Heinzen et al., 2010). Additional genetic variation might explain the phenotypic variability of all three microdeletions as suggested by a significantly higher burden of large heterozygous deletions in epilepsy patients compared to controls (Heinzen et al., 2010). Within this study, we concentrated on known, recurrent microdeletions. Therefore, we cannot comment on the overall CNV load including rare copy number variations in the cohort. The frequency of additional, rare CNVs might be higher as indicated by previous studies (Mefford et al., 2010, 2011).
Conclusions In conclusion, in our pediatric cohort, recurrent microdeletions at 15q11.2, 15q13.3 and 16p13.11 are mainly associated with phenotypes of the spectrum of idiopathic generalized epilepsies or related generalized EEG patterns. In contrast to previous reports, these recurrent microdeletions are virtually absent in focal epilepsies, FS, FS+ and GEFS+. Microdeletion carriers have a five-fold risk to present with various degrees of ID compared to patients without these risk factors. This microdeletion triad might help delineate a novel spectrum of epilepsy phenotypes classifiable through clinical, electrographic and genetic data.
Acknowledgements The authors would like to thank the patients and their parents for participation in this study. We are grateful to Angelika Ackerhans and Klaus Moldenhauer for database management and Anita Dietsch, Catharina Fürstenau, Katja Schmidt-Cloppenborg, Ilona Urbach and Tanja Wesse for technical assistance.
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