Journal of Child Neurology

Journal http://jcn.sagepub.com/ of Child Neurology

Copy Number Variations in Patients With Electrical Status Epilepticus in Sleep Sietske H.G. Kevelam, Floor E. Jansen, Ellen van Binsbergen, Kees P.J. Braun, Nienke E. Verbeek, Dick Lindhout, Martin Poot and Eva H. Brilstra J Child Neurol 2012 27: 178 originally published online 27 September 2011 DOI: 10.1177/0883073811416006 The online version of this article can be found at: http://jcn.sagepub.com/content/27/2/178

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Original Article

Copy Number Variations in Patients With Electrical Status Epilepticus in Sleep

Journal of Child Neurology 27(2) 178-182 ª The Author(s) 2012 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0883073811416006 http://jcn.sagepub.com

Sietske H.G. Kevelam, MD1, Floor E. Jansen, MD, PhD2, Ellen van Binsbergen, MSc1, Kees P.J. Braun, MD, PhD2, Nienke E. Verbeek, MSc, MD1, Dick Lindhout, MD, PhD1, Martin Poot, PhD dr. med. habil.1, and Eva H. Brilstra, MD, PhD1

Abstract Electrical status epilepticus in sleep syndrome is the association of the electroencephalographic pattern and deficits in language or global cognitive function and behavioral problems. The etiology is often unknown, but genetic risk factors have been implicated. Array-based comparative genomic hybridization was used to identify copy number variations in 13 children with electrical status epilepticus in sleep syndrome to identify possible underlying risk factors. Seven copy number variations were detected in 4 of the 13 patients, which consisted of 6 novel gains and 1 loss, the recurrent 15q13.3 microdeletion. Two patients carried a probable pathogenic copy number variation containing a gene involved in the cholinergic pathway. Genetic aberrations in patients with electrical status epilepticus in sleep syndrome can provide an entry in the investigation of the etiology of electrical status epilepticus in sleep. However, further studies are needed to confirm our findings. Keywords electrical status epilepticus in sleep, copy number variation, array-based comparative genomic hybridization Received May 22, 2011. Received revised June 9, 2011. Accepted for publication June 11, 2011.

Electrical status epilepticus in sleep refers to an electroencephalographic pattern of continuous, subclinical sleep-induced spike-and-waves.1 Electrical status epilepticus in sleep is predominantly seen in Landau-Kleffner syndrome, the syndrome of continuous spike-and-waves during sleep and in some children with atypical benign focal epilepsy of childhood.2 These syndromes share an association of seizures, deficits in language or global cognitive functioning and behavioral problems. In most patients, the etiology remains unknown.2 However, genetic etiologies have been identified in related disorders with overlapping clinical features. In patients with benign childhood epilepsy with centro-temporal spikes, with speech and language problems, mutations in the SRPX2 gene have been found.3 Furthermore, an SRPX2 mutation was identified in a patient with bilateral perisylvian polymicrogyria, a malformation of cortical development that has been associated with electrical status epilepticus in sleep.3 Another patient with electrical status epilepticus in sleep, cognitive decline, and refractory epilepsy carried a neuroserpin gene mutation.4 These findings implicate that genetic factors can play a role also in the pathogenesis of electrical status epilepticus in sleep. With array-based comparative genomic hybridization technologies, submicroscopic genomic losses and gains, referred to as copy number variations, can be identified.5 Array-based comparative genomic hybridization technology is a relatively

new technique that has emerged as a diagnostic tool to detect copy number variations in the genome that possibly are not detectable by conventional cytogenetic methods, including high-resolution chromosomal analysis or fluorescence in situ hybridization studies. The implementation of array-based comparative genomic hybridization technology has led to the identification of novel genomic disorders in patients with mental retardation, which allows for proper genetic counseling and increases insights in the pathogenesis of these disorders.6,7 Recent studies have demonstrated that specific recurrent copy number variations are overrepresented among patients with idiopathic generalized epilepsy and point to specific molecular pathways.8-10 Recurrent copy number variations as well as novel copy number variations, if identified in patients with

1

Department of Medical Genetics, University Medical Center Utrecht, the Netherlands 2 Department of Child Neurology, Rudolf Magnus Institute of Neuroscience, University Medical Center Utrecht, the Netherlands Corresponding Author: Sietske H.G. Kevelam, Department of Medical Genetics, Division of Biomedical Genetics, University Medical Center Utrecht, P.O. Box 85090, 3508 AB Utrecht, the Netherlands Email: [email protected]


Kevelam et al

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Table 1. Results of Array-based Comparative Genomic Hybridization of the Four Patients With Copy Number Variations Patient Number

Loss/ Gain

Origin

Chromosome

Start- Endb

Genes located within the copy number variation

1

Loss

De novo

15q13.3

29.000.000-30.400.000

2

Gain Gain Gain Gain Gain Gain

Maternala Paternal Maternal Unknown Unknown Unknown

Xp22.11 9q34.3 Xp22.33 5p12 5q31.3 16q23.1

24.270.000-24.760.000 138.150,000-138.380.000 225.426-690.299 43.820.000-44.380.000 141.970.000-142.430.000 75.750.000-76.220.000

ARHAGAB11B, MIMK15, MIMR10, TRPM1, KLF13, OTUD7A, CHRNA7c PDK3, PCYT1Bc, POLA1 LHX, QSOX2, GPSM1, CORF151, CARD9 SHOX, part of PPP2R3B Part of FGF10 Part of FGF1, ARHGAP26 ADAMTS18, MON1B

3 4

a

Skewed X chromosome inactivation of more than 99%. Minimally deleted or duplicated region. c Genes involved in the cholinergic pathway. b

electrical status epilepticus in sleep syndrome, can improve our insights into possible genetic factors that can contribute to the epileptiform activity in sleep and the cognitive deficits. In this study, we performed array-based comparative genomic hybridization analysis in 13 patients with electrical status epilepticus in sleep syndrome to detect copy number variations to identify possible underlying genetic mechanisms.

encompassed at least 3 consecutive spots on the array were conserved. Interpretation of copy number variation data was performed as described in the guidelines of Vermeesch et al.11 Copy number variations that are considered polymorphisms according to these guidelines were not reported. X-inactivation pattern analysis was performed by studying the methylation of the HhaI sites in the first exon of the human androgen receptor locus as previously reported.12

Data Analysis

Patients and Methods Patients We consecutively included all patients diagnosed with electrical status epilepticus in sleep syndrome between January 2000 and August 2009 at the Pediatric Neurology outpatient Clinic of the University Medical Center Utrecht. Electrical status epilepticus in sleep was defined as a significant increase of spike-waves during sleep (spike-wave index > 75%), unilateral or bilateral, assessed in multiple sleep electroencephalograms. Patients with epileptic encephalopathy, including West Syndrome and Lennox-Gastaut syndrome, were excluded. In none of these patients a specific metabolic, structural, or genetic etiological diagnosis had been made, so array-based comparative genomic hybridization analysis was considered indicated. A detailed patient and family history was taken with respect to epilepsy characteristics, mental development, speech and language problems, and features of autism. Additional clinical data were extracted from medical records. All patients had undergone a brain magnetic resonance imaging (MRI) and most patients had multiple neuropsychological examinations. Informed consent for data collection and array-based comparative genomic hybridization analysis was obtained.

Genetic Analysis Genomic DNA was isolated from peripheral blood using standard protocols. Microarray-based comparative genomic hybridization analysis was performed in a diagnostic setting using 105K (amadid 019015) until July 2009 or 180K (amadid 023363) from July 2009 microarray slides, from Agilent Technologies (Santa Clara, CA) and following manufacturer’s protocols. Scanned images were analyzed using the Feature extraction software (Agilent Technologies). Data were analyzed using DNA analytics 4.76 software from Agilent Technologies using the Aberration Detection Method 2 (ADM-2) algorithm with the following settings: a call consists of minimally 3 consecutive probes with a log ratio of + 0.3. Therefore, only gains or losses that

To gain more insight into the potential pathogenic contribution of the identified copy number variations, we studied their mode of inheritance, and the expression pattern and function of the genes located within the copy number variations. In addition, we reviewed the literature and searched in databases for similar copy number variations in patients. We used the Mann-Whitney U test and the Fisher’s exact test to compare the clinical characteristics of patients with copy number variations on the one hand, and patients without copy number variations on the other.

Results Twenty patients with electrical status epilepticus in sleep syndrome were identified. Three patients were lost to follow-up, and informed consent was not received for 4 others. Arraybased comparative genomic hybridization analysis was available on DNA samples from 13 patients. Based on the clinical characteristics and the electroencephalographic pattern 5 of 13 patients were diagnosed with Landau-Kleffner syndrome, 4 with continuous spike-and-waves during sleep, and 4 with atypical benign focal epilepsy of childhood. Seven copy number variations were identified in 4 patients, which consisted of 6 novel gains and 1 loss, the recurrent 15q13.3 microdeletion (Table 1). The copy number variation in patient 1 was de novo. Two copy number variations (patient 2 and 3) were maternally inherited. The X-chromosomal gain in patient 2 is located outside de pseudo-autosomal dominant region. The X-inactivation pattern analysis in the mother showed skewed X-inactivation of more than 99%. The X-chromosomal gain in patient 3, which was also maternally inherited, is located in the pseudoautosomal region. The mother had not been diagnosed with epilepsy, cognitive impairment, or a behavioral disorder.


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Journal of Child Neurology 27(2)

Table 2. Clinical Characteristics of the Patients with Copy Number Variations Patient Early no. development

Age of diagnosis (years) Cognitive deficita

Intelligence test at Behavioral diagnosis problemsb

1

Normal

6,9

Global regression

56

2

Speech and language delay Normal

7,6

Global regression