GRIN2A in common epilepsies / 1
Investigation of GRIN2A in common epilepsy phenotypes
Dennis Lal1, PhD, Sandra Steinbrücker2, Julian Schubert3, Thomas Sander1, MD, Felicitas Becker3, Yvonne Weber3, Holger Lerche3, MD, Holger Thiele1, PhD, Roland Krause4, Anna-Elina Lehesjoki5, Peter Nürnberg1,6, Bernd A. Neubauer7, MD, Hiltrud Muhle8, Ulrich Stephani8, Ingo Helbig8,9, MD, Albert Becker10, PhD, Susanne Schoch10, Jörg Hansen11, MD, Thomas, Dorn11, MD, Christin Hohl2, Nicole Lüscher2, Epicure consortium§, EuroEPINOMICS-CoGIE consortium§§, Sarah von Spiczak5,12,*,#, MD, Johannes R. Lemke2,13,*, MD
Affiliations
1 Cologne Center for Genomics, University of Cologne, Cologne, Germany
2 Division of Human Genetics, University Children's Hospital Inselspital, Bern, Switzerland
3 Department of Neurology and Epileptology, Hertie Institute of Clinical Brain Research, University of Tübingen, Tübingen, Germany
4 Luxembourg Centre for Systems Biomedicine, Belval, Luxembourg
5Neuroscience Center, University of Helsinki and Folkhälsan Institute of Genetics, Helsinki, Finland
6Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
7Department of Neuropediatrics, University Medical Faculty Giessen and Marburg, Giessen, Germany
8Department of Neuropediatrics, University Medical Center Schleswig-Holstein, Kiel, Germany
9Children’s Hospital of Philadelphia (CHOP), Philadelphia, Pennsylvania, USA
10Department of Neuropathology, University of Bonn Medical Center, Bonn, Germany
11 Swiss Epilepsy Center, Zürich, Switzerland
12 Northern German Epilepsy Center for Children and Adolescents, Schwetinental-Raisdorf, Germany
13 Institute of Human Genetics, University Medical Center Leipzig, Leipzig, Germany
§ EPICURE consortium = EPICURE Integrated Project (contributing centers listed by country): Department of Clinical Neurology (Fritz Zimprich) and Department of Pediatrics and Neonatology (Martina Mörzinger, Martha Feucht), Medical University of Vienna, 1090 Vienna, Austria. VIB Department of Molecular Genetics, University of Antwerp, 2610 Antwerpen, Belgium (Arvid Suls, Sarah Weckhuysen, Lieve Claes, Liesbet Deprez, Katrien Smets, Tine Van Dyck, Tine Deconinck, Peter De Jonghe). Research Department, Danish Epilepsy Centre, 4293 Dianalund, Denmark (Rikke S Møller, Laura L. Klitten, Helle Hjalgrim). Wilhelm Johannsen Centre for Functional Genome Research, University of Copenhagen, 2200 Copenhagen N, Denmark (Rikke S Møller). Department of Neuropediatrics, University Medical Center Schleswig-Holstein (Kiel Campus), 24105 Kiel, Germany (Ingo Helbig, Hiltrud Muhle, Philipp Ostertag, Sarah von Spiczak, Ulrich Stephani). Cologne Center for Genomics, University of Cologne, 50931 Cologne, Germany (Peter Nürnberg, Thomas Sander, Holger Trucks). Department of Epileptology, University Clinics Bonn, 53105 Bonn, Germany (Christian E. Elger, Ailing A. Kleefuß-Lie, Wolfram S. Kunz, Rainer Surges). Department of Neurology, Charité University Medicine, Campus Virchow Clinic, Humboldt University of Berlin, 13353 Berlin, Germany (Verena Gaus, Dieter Janz, Thomas Sander, Bettina Schmitz). Epilepsy-Center Hessen, Department of Neurology, Philipps-University Marburg, 35043 Marburg, Germany (Karl Martin Klein, Philipp S. Reif, Wolfgang H. Oertel, Hajo M. Hamer, Felix Rosenow). Abteilung Neurologie mit Schwerpunkt Epileptologie, Hertie Institut für klinische Hirnforschung, Universität Tübingen, 72076 Tübingen, Germany (Felicitas Becker, Yvonne Weber, Holger Lerche). Department of Neurology, University of Munich Hospital – Großhadern, Munich, Germany (Claudia Kapser, Christoph J. Schankin). Netherlands Section Complex Genetics, Department of Medical Genetics, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands (Bobby P.C. Koeleman, Carolien de Kovel, Dick Lindhout). SEIN Epilepsy Institute in the Netherlands, 2130AM Hoofddorp, Netherlands (Dick Lindhout).
§§EuroEPINOMICS-CoGIE consortium(contributing centers listed by country): Department of Neurology (Eva M. Reinthaler, Fritz Zimprich) and Department of Pediatrics and Neonatology (Martha Feucht), Medical University of Vienna, 1090 Vienna; Private Practice for Pediatrics (Hannelore Steinboeck), 1150Vienna; St. Anna Children’s Hospital, Department of Neuropediatrics (Birgit Neophytou), 1090 Vienna; Department of Pediatrics (Julia Geldner), Hospital SMZ Sued Kaiser-Franz-Josef, 1100 Vienna; Department of Pediatrics (Ursula Gruber-Sedlmayr), Medical University of Graz, 8036 Graz; Department of Pediatrics (Edda Haberlandt), Medical University of Innsbruck, 6020 Innsbruck, Austria. Department of Pediatrics (Gabriel M. Ronen), McMaster University, L8N3Z5 Hamilton, Ontario, Canada. Cologne Center for Genomics (Janine Altmueller, Dennis Lal, Peter Nuernberg, Thomas Sander, Holger Thiele) University of Cologne, 50931 Cologne; Department of Neurology and Epileptology, Hertie Institute of Clinical Brain Research (Felicitas Becker, Holger Lerche, Yvonne Weber), University of Tuebingen, 72076 Tuebingen; Department of Neuropediatrics (Bernd Neubauer), University Medical Faculty Giessen and Marburg, 35385 Giessen, Germany.
Corresponding author
Sarah von Spiczak, MD, Northern German Epilepsy Center for Children and Adolescents, Henry-Dunant-Str. 6-8, 24223 Schwentinental-Raisdorf, Germany
Phone: +49 4307 909 201; Fax: +49 4307 909 260; E-Mail:
Running title GRIN2A in common epilepsies
Key wordsGRIN2A, idiopathic generalized epilepsy, temporal lobe epilepsy, mutation, copy number variation
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Summary
Recently, mutations and deletions in the GRIN2A gene have been identified to predispose to benign and severe idiopathic focal epilepsies (IFE), revealing a higher incidence of GRIN2A alterations among the more severe phenotypes. This study aimed to explore the phenotypic boundaries of GRIN2A mutations by investigating patients with the two most common epilepsy syndromes: i) idiopathic generalized epilepsy (IGE) and ii) temporal lobe epilepsy (TLE). Whole exome sequencing data of 238 patients with IGE as well as Sanger sequencing of 84 patients with TLE were evaluated for GRIN2A sequence alterations. Additionally, two independent cohorts comprising 1469 IGE and 330 TLE patients were screened for structural deletions (>40 kb) involving GRIN2A. Apart from a presumably benign, non-segregating variant in a patient with juvenile absence epilepsy, neither mutations nor deletions were detected in either cohort. These findings suggest that mutations in GRIN2A preferentially are involved in genetic variance of pediatric IFE and do not contribute significantly to neither adult focal epilepsies as TLE nor generalized epilepsies.
Introduction
Mutations in GRIN2A, coding for the alpha2 subunit of the NMDA glutamate receptor, were recently identified in pediatric patients with epilepsy-aphasia spectrum disorders including benign focal epilepsies with centrotemporal spikes (CTS)(Carvill et al., 2013, Lemke et al., 2013, Lesca et al., 2013). Most patients were described with epileptic phenotypes belonging to the more severe end of the spectrum of idiopathic focal epilepsies (IFE) such as atypical Rolandic epilepsy, atypical benign partial epilepsy (Pseudo-Lennox syndrome, ABPE), epileptic aphasia (Landau-Kleffner syndrome, LKS) and electrical status epilepticus in slow-wave sleep (ESES). Mutation frequency ranged from 4.9% in Rolandic epilepsy to 17.6% in Landau-Kleffner syndrome(Lemke et al., 2013). Exon-disrupting copy number variations(CNV) in GRIN2A occurred in 1% of patients with pediatric IFE(Lemke et al., 2013). In line with this observation, previous studies reported GRIN2A mutations as well as deletions in different epilepsy disorders associated with CTS on EEG (Endele et al., 2010, Reutlinger et al., 2010).
The aim of this study was to further delineate the phenotypic spectrum of GRIN2A mutations and deletions by performing a genetic screen of patients with idiopathic generalized epilepsy (IGE) and temporal lobe epilepsy (TLE).
Methods
This study has been approved by the local Research Ethics Committees. All study participants provided an informed written consent. Phenotyping was performed according to the 2001 and 2010 International League Against Epilepsy (ILAE) classification schemes(Engel, 2001, Berg et al., 2010). Both investigated epilepsy syndromes, IGE and TLE, were not further subdivided into different subtypes. The targeted sequencing data for identification of single nucleotide variants (SNV) as well as the SNP-array data for identification of structural deletions (>40 kb) were analyzed for genomic alterations affecting the GRIN2A gene (chr16:9,847,265-10,276,263; human genome build 37/hg19). Of note, due to limitations in the accuracy of structural duplication calling, we have excluded duplications from the analysis. A summary of all investigated cohorts is provided in table 1.
DNA-extraction from blood samples
DNA from individual blood samples was extracted locally at the recruitment centers using commercially available kits and standard methods.
IGE: exome sequencing
Sequence analysis was performed in 238 unrelated patients with IGE of self-identified European ancestry using next generation sequencing techniques. DNA was fragmented using sonification technology (Covaris, Woburn, MA, USA) and fragments were end repaired and adaptor ligated. Enrichment was performed using the SeqCap EZ Human Exome Library® v2.0 (Roche NimbleGen, Madison, WI, USA) and analysis of samples was done on the IlluminaHiSeq 2000® sequencer (Illumina, San Diego, CA, USA). Inclusion criteria for data analysis were defined as follows: average coverage >30x for 85% of the target sequences. Data filtering was done using Illumina Realtime Analysis® (RTA) software v1.8 followed by mapping to the human genome reference build hg19 via the ELANDv2 alignment algorithm. CASAVA v1.8 was used to exclude PCR duplicates. Variant calling was performed by SAMtools (version 0.1.7) for InDel detection. For the detection of protein changes, affected splice sites, and overlaps with known variants,in-house scripts, developed at the Cologne Center for Genomics (Cologne, Germany), were used.
IGE: Structural deletion analysis
Deletion screening was carried out in 1469 unrelated patients with IGE of self-identified North-Western European ancestry (for details see(Lal et al., 2013)). DNA samples were genotyped with the Affymetrix Genome-Wide Human SNP Array 6.0 (Affymetrix, Santa Clara, CA, USA). Deletion calling was performed, using the Birdsuit algorithm implemented in the Affymetrix Genotyping Console version 4.1.1. Deletion analysis was restricted to deletions larger than 40 kb covering at least 20 consecutive probes. Regional log2 ratios of the signal intensities and the SNP heterozygosity state were visualized and manually inspected in the Chromosome Analysis Suite v1.2.2 (Affymetrix, Santa Clara, CA, USA).
TLE: Sanger sequencing of GRIN2A
Sequence analysis was performed in 84 unrelated patients with mixed TLE phenotypes of self-identified European ancestry using routine Sanger sequencing techniques for all 13 exons and intron-exon boundaries of GRIN2A with the BigDye Terminator v3.1 Cycle Sequencing kit on an ABI3730 DNA Analyser (Applied Biosystems, Foster City, CA). Primers and PCR conditions are available upon request.
TLE: Structural deletion analysis
DNA of 330 patients with mesial temporal lobe epilepsy was assayed with the Infinium II Sentrix HumanHap550v3 duo Genotyping BeadChip (Illumina, San Diego, CA, USA), containing over 550,000 unique tag SNP markers. Hybridizations were performed according to the manufacturer’s instructions. Analysis of genotypes was done with the Illumina Genome Studio® genotyping module (v.2011). The PennCNV software was used to generate deletion calls by calculating the log R ratio (LRR) and B allele frequency (BAF) for all markers. Only deletions larger than 40 kb and covering at least 20 consecutive probes were analyzed. Technical artifacts were excluded by manual inspection of the regional SNP heterozygosity state and log2 ratios of the signal intensities.
Mutation screening
Data were filtered for unknown variants in GRIN2A and compared to an in-house variation database, dbSNP build 135, 1000Genomes database and the Exome Variant Server. To assess possible pathogenic implications of identified variants, several in silico analysis programs were used (PolyPhen2 and MutationTaster for coding variants, SpliceView and HSF2.4 for SNVs at exon boundaries/ potential splice sites).
Results
Idiopathic generalized epilepsy
238 patients with idiopathic generalized epilepsy were screened for mutations in the coding sequence of GRIN2A. Only one novel non-synonymous variant (c.4135A>G, p.I1379V; NM_001134407.1) was identified in a girl with juvenile absence epilepsy, exhibiting sporadic absences and generalized tonic-clonic seizures on awakening. This variant was rated as benign by prediction programs and did not segregate with the phenotype in the family (Fig.1).
In addition, SNP array data of 1469 patients with IGE were analyzed for structural deletions. Neither exon-disrupting nor larger deletions covering GRIN2A were detected in this cohort.
Figure 1
Temporal lobe epilepsy
Sanger sequencing of GRIN2A in 84 patients with TLE did not reveal previously unknown sequence alterations. Only three patients carried one single coding missense variant each, all of which were previously described in public databases: c.422C>T, p.T141M (rs78631453); c.2627T>C, p.I876T (rs199784503); c.2899G>C, p.V967L (rs61731465).
SNP array data of 330 patients with temporal lobe epilepsy were analyzed for genetic alterations in GRIN2A. No deletion affecting the genomic sequence of GRIN2A was detected.
Discussion
The first publication reporting GRIN2A mutations in patients with epilepsy described four mutation carriers in two families, one index patient presenting with moderate developmental delay and CTS, the other with a more complex phenotype of epileptic encephalopathy with epileptic spasms, myoclonic seizures, generalized slowing as well as multifocal spikes on the EEG and profound developmental delay (Endele et al., 2010). Recently, three large collaborative studies (Carvill et al., 2013, Lemke et al., 2013, Lesca et al., 2013) identified mutations in GRIN2A as an important risk factor for pediatric patients expressing IFE with the hallmark of CTS. Mutations were more frequent in the severe phenotypes of the spectrum, namely ABPE, LKS and ESES as well as in atypical IFE, belonging to the wider spectrum of the epilepsy aphasia spectrum. Since then, several patients with GRIN2A aberrations have been described. In the majority of cases (Dimassi et al., 2014, Conroy et al., 2014) the respective phenotypes were within the spectrum described previously(Endele et al., 2010, Reutlinger et al., 2010, Carvill et al., 2013, Lemke et al., 2013, Lesca et al., 2013). However, additional case reports were published describing mutations also in less defined and more severe phenotypes similar to the second patient described by Endele and colleagues(Venkateswaran et al., 2014, DeVries and Patel, 2014, Pierson et al., 2014). These reports indicate that genetic alterations of GRIN2A are not limited to IFE but rather predispose to a broader range of epilepsy syndromes with benign IFE at the one end and unclassified epilepsy as well as epileptic encephalopathy and severe developmental disorders at the other. Besides this pleiotropic expression, inherited GRIN2A alterationsdisplay incomplete penetrance(Carvill et al., 2013, Lemke et al., 2013, Lesca et al., 2013). The individual disease phenotype is probably further specified by the interplay with genetic background effects and environmental influences following an oligo-/polygenic inheritance model with genetic heterogeneity(Dibbens et al., 2007).
In agreement with this model, pleiotropy of epilepsy genes has already been described for classical epilepsy genes such as SCN1A, PCDH19, KCNQ2 and several others. A striking example for an unexpected underlying shared genetic disease etiology for clinical diverse epilepsies is depicted by the DEPDC5 gene. Recently, several studies were published on mutations in DEPDC5 linking various forms of focal epilepsies to a common genetic origin (Poduri, 2014). The described spectrum comprises IFE (Lal et al., 2014) as well as other forms of familial and sporadic focal epilepsies including temporal and frontal lobe epilepsies (Ishida et al., 2013, Dibbens et al., 2013). In addition, even patients with symptomatic epilepsies caused by malformations were identified as mutation carriers (Scheffer et al., 2014).
Here, we investigated the occurrence of GRIN2A mutations and CNVs in two forms of common epilepsies, i.e. IGE and TLE. We failed to identify any obvious pathogenic mutations or deletions. These findings suggest that genetic alterations in GRIN2A contribute to a spectrum of diseases ranging from benign to severe and from well-defined phenotypes to unclassified epilepsies similar to other epilepsy genes such as PCDH19 and KCNQ2 but do not cause a wider spectrum of pediatric and adult focal epilepsies such as DEPDC5. Further studies are needed to more precisely define the phenotypic boundaries of epilepsies associated with GRIN2A and other glutamate receptor genes.
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Acknowledgements
We thank all patients and family members for their participation in this study. Furthermore, we are grateful to all clinicians referring patients and probands for genetic research. We would like to thank all lab technicians for technical assistance with mutation and CNV analysis.
J.R.L. (32EP30_136042 / 1) and I.H. (HE5415/3-1) received financial support within the EuroEPINOMICS-RES network and H.L., B.N., P.N., T.S., and F.Z. received financial support within the EuroEPINOMICS-CoGIE network(DFG grant numbers: HL: LE1030/11-1, BN: BN416/5-1, PN: NU50/8-1, TS: SA434/5-1, FWF grant number: FZ: I643-B09) and T.S. was supported by the EuroEPINOMICS-EpiGENet network (SA434/5-1), all within the EUROCORES framework of the European Science Foundation (ESF, In addition, this work was supported by grants from the European Community [FP6 Integrated Project EPICURE, grant LSHM-CT-2006-037315 to B.P.C.K., H.L., and T.S.]and the German Federal Ministry of Education and Research, National Genome Research Network [NGFNplus: EMINet, grants 01GS08120 to T.S., and 01GS08123 to H.L.];
Disclosure of Conflicts of Interest
None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.
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