Multiple rare variants in Rolandic epilepsy

Rationale: Rolandic epilepsy (RE), also known as benign childhood epilepsy with centrotemporal spikes, is the most common form of idiopathic epilepsy in childhood with a strong underlying genetic aetiology (presumably oligo- to polygenetic). RE is an attractive object for genetic studies because of its shared genetic background with other forms of epilepsies (e.g. locus 15q13.3). Moreover, its high prevalence amongst school-aged children and the typical clinical manifestation allow the easy and unequivocal recruitment of sufficient sample numbers Recent considerations on the inheritance of common diseases have stressed the likely importance of multiple rare variants (variants below a population frequency of ~5%). Specifically, multiple rare variants occurring on multiple loci (per disease) have been put forward as an explanation for the "missing heritability" observed in many common diseases (i.e. the sometimes surprising negativity of linkage analyses and the failure of common genetic variants to account for the existing high heritability). If multiple rare variants on a restricted number of genes are indeed responsible to a relevant degree for common diseases, sequencing the whole exome (i.e. the whole of the protein coding regions) in a sufficient number of affected individuals should be a promising strategy. In a recent study the potential success of this "gene-finding" method has been demonstrated. Objectives: The goal of this study is to identify rare sequence variations relevant for the pathogenesis of RE using next generation whole exome sequencing. Recruitment: Within the CoGIE-network we already have access to 120 multiplex families with RE, over 50 parent-offspring trios and 200 sporadic cases. These numbers are sufficient for the initial phases of the project, but to ensure the necessary higher sample numbers for the confirmatory sequencing and genotyping (IP3 and IP4) the first focus of this proposal will be to recruit further patients (goal >375 sporadic casesrios) through our international and national networks. Whole exome sequencing: We plan to capture and sequence the exomes of 25 individuals with familial Rolandic epilepsy (Illumina GA-platform). The exomes of a further 60 patients with familial RE will be sequenced by the investigators of AP1 (Palotie, Sanger Institute), so in total the sequences of the whole protein coding regions of 85 patients with RE will be available for further analysis. This process together with the efforts of IP3 (WGA in RE) and with the bioinformatics input of (IP5) will define candidate genes (i.e. genes with a high mutational load and/or strongly associated SNPs) Confirmation stage: Candidate genes and identified mutations will then need to be confirmed by resequencing and genotyping in a larger sample of RE patients and controls. Given the current sequencing costs and the budget limitations we plan to sequence the 10 most interesting genes by conventional capillary sequencing methods (in 192 RE patients). Should the costs of targeted next generation sequencing fall we would employ this method to resequence more candidate genes. Presumed pathogenic variants will be further evaluated by genotyping more patients and controls (MALDI-TOF platform, ~90 SNPs, 768 individuals) and by performing co-segregation in all familial cases. Contribution of IP4 to the CoGIE CRP: In this IP we focus on the contribution of rare variants with a large effect size to the aetiology of Rolandic epilepsy. Our work will complement (i) the efforts of IP3 which investigates the role of common genetic variants and of copy number variations to the pathogenesis of RE and (ii) the parallel efforts of other groups working on the genetics of IGEs (AP1, AP2, IP1, IP3). One interactive point will be to evaluate if both most common forms of idiopathic epilepsies - RE and IGE - have an overlapping genetic basis, since this is not obvious from familial clinical data but may be suggested from some molecular genetic findings (Ref 1,5). Furthermore, IP4 will closely interact with AP1, IP3 and IP5 for bioinformatics analysis and integration of the GWAS and sequencing data. Finally, the most interesting sequence variants will be the basis for functional investigations in projects IP6-7, AP5-7.

The main aim of the project "multiple rare variants in rolandic epilepsy" was the elucidation of the genetic background of the most common childhood epilepsies (so called idiopathic focal epilepsies or short IFE). The range of theses epilepsies comprises benign variants as rolandic epilepsy but also severe epileptic encephalopathies with additional neuropsychiatric symptoms such as the Landau-Kleffner syndrome. At the start of the project a genetic basis for IFE was assumed by many (but not all) experts but little concrete knowledge was available about the underlying genes. Within this international project we managed to recruit over 250 families with IFE from Austria, Germany and Canada to perform state of the art genetic analyses (e.g. exome sequencing). By employing sophisticated analysis-techniques we could search for causative genetic variants for IFE. Eventually, for the first time genes for these epilepsies could be unequivocally identified. One important contribution was the identification of the gene GRIN2A as the most frequent genetic cause for idiopathic childhood focal epilepsies, approximately 7,5% of all IFE, especially severe variants, are caused by mutations in this gene (Lemke et al. 2013). This gene codes for an NMDA receptor-subunit, an important molecule for learning-processes. Given these parallels a close relationship between epilepsies and learning can be discussed. An another important insight was the finding of duplications on chromosome 16p11.2 (a stretch of the DNA is repeated) as a clear cause of IFEs (Reinthaler et al. 2014). This is particularly interesting, because several neuropsychiatric disorders such as autism can be caused by the same duplication, which suggests common mechanisms for both diseases. In addition to the above mentioned findings a range of other genes could be implicated as causes for IFE. We could for example identify mutations in the receptors for the neurotransmitter GABA as well as in the genes DEPDC5 and RBFOX3 as being responsible for IFE. As these genes have already been described in other forms of epilepsies the common genetic causes between childhood focal epilepsies and other seizure disorders could been documented (Lal et al. 2013, 2014, Reinthaler et al. 2014, 2015).In conclusion, in this project, we could for the first time unequivocally identify genetic causes for these common childhood epilepsies. These findings are an important contribution to our understanding of the mechanisms for these disorders and enable us to perform individual diagnostic investigations.