Bernd A. Neubauer

Mutations in GRIN2A cause idiopathic focal epilepsy with rolandic spikes

Idiopathic focal epilepsy (IFE) with rolandic spikes is the most common childhood epilepsy, comprising a phenotypic spectrum from rolandic epilepsy (also benign epilepsy with centrotemporal spikes, BECTS) to atypical benign partial epilepsy (ABPE), Landau-Kleffner syndrome (LKS) and epileptic encephalopathy with continuous spike and waves during slow-wave sleep (CSWS). The genetic basis is largely unknown. We detected new heterozygous mutations in GRIN2A in 27 of 359 affected individuals from 2 independent cohorts with IFE (7.5%; P = 4.83 × 10−18, Fishers exact test). Mutations occurred significantly more frequently in the more severe phenotypes, with mutation detection rates ranging from 12/245 (4.9%) in individuals with BECTS to 9/51 (17.6%) in individuals with CSWS (P = 0.009, Cochran-Armitage test for trend). In addition, exon-disrupting microdeletions were found in 3 of 286 individuals (1.0%; P = 0.004, Fishers exact test). These results establish alterations of the gene encoding the NMDA receptor NR2A subunit as a major genetic risk factor for IFE.

Genetic dissection of photosensitivity and its relation to idiopathic generalized epilepsy

Photosensitivity or photoparoxysmal response (PPR) is a common and highly heritable electroencephalographic trait characterized by an abnormal visual sensitivity of the brain in reaction to intermittent photic stimulation. PPR occurs frequently associated with idiopathic generalized epilepsies (IGEs). The present genomewide linkage scan was designed to map susceptibility loci for PPR and to explore their genetic relationship with IGE. The study included 60 families with at least two siblings displaying PPR. To dissect PPR‐specific and IGE‐related susceptibility loci, we defined two distinct family subgroups, comprising 19 families with predominantly pure PPR and photosensitive seizures (PPR‐families) and 25 families, in which PPR was strongly associated with IGE (PPR/IGE‐families). MOD score analyses provided significant evidence for linkage to the region 6p21.2 in the PPR‐families (empirical p = 0.00004) and suggestive evidence for linkage to the region 13q31.3 in the PPR/IGE families (p = 0.00015), both with a best‐fitting recessive mode of inheritance. In the PPR/IGE‐families, linkage evidence was even stronger (p = 0.00003) when the trait definition was broadened by IGE traits. Our study shows two PPR‐related susceptibility loci, depending on the familial background of IGE. The locus on 6p21.2 seems to predispose to PPR itself, whereas the locus on 13q31.3 also confers susceptibility to IGE. Ann Neurol 2005;57:866–873

PRRT2 Mutations are the major cause of benign familial infantile seizures

Mutations in PRRT2 have been described in paroxysmal kinesigenic dyskinesia (PKD) and infantile convulsions with choreoathetosis (PKD with infantile seizures), and recently also in some families with benign familial infantile seizures (BFIS) alone. We analyzed PRRT2 in 49 families and three sporadic cases with BFIS only of Italian, German, Turkish, and Japanese origin and identified the previously described mutation c.649dupC in an unstable series of nine cytosines to occur in 39 of our families and one sporadic case (77% of index cases). Furthermore, three novel mutations were found in three other families, whereas 17% of our index cases did not show PRRT2 mutations, including a large family with late‐onset BFIS and febrile seizures. Our study further establishes PRRT2 as the major gene for BFIS alone. Hum Mutat 33:1439–1443, 2012.

Children with Focal Sharp Waves: Clinical and Genetic Aspects

Summary: Purpose: To investigate the spectrum of clinical manifestations in children with benign focal sharp waves in the EEG to gain further insight into the genetic background of clinical and EEG symptomatology in a family study.

KCNQ2 and KCNQ3 mutations contribute to different idiopathic epilepsy syndromes

Objective: To explore the involvement of M-type potassium channels KCNQ2, Q3, and Q5 in the pathogenesis of common idiopathic epilepsies. Methods: Sequence analysis of the KCNQ2, Q3, and Q5 coding regions was performed in a screening sample consisting of 58 nuclear families with rolandic epilepsy. Subsequently, an association study was conducted for all discovered variants in a case–control sample comprising 459 German patients with idiopathic generalized epilepsy (IGE) and 462 population controls. Results: An in-frame deletion of codon 116 in KCNQ2 (p.Lys116del) and a missense mutation in KCNQ3 (p.Glu299Lys) were detected in two index cases exhibiting rolandic epilepsy and benign neonatal convulsions. Both mutations resulted in reduced potassium current amplitude in Xenopus oocytes. Mutation analysis of families with rolandic epilepsy without neonatal seizures discovered three novel missense variations (KCNQ2 p.Ile592Met, KCNQ3 p.Ala381Val, KCNQ3 p.Pro574Ser). The KCNQ2 p.Ile592Met variant displayed a significant reduction of potassium current amplitude in Xenopus oocytes and was present only once in 552 controls. Both missense variants identified in KCNQ3 (p.Ala381Val and p.Pro574Ser) were present in all affected family members and did not occur in controls, but did not show obvious functional abnormalities. The KCNQ3 missense variant p.Pro574Ser was also detected in 8 of 455 IGE patients but not in 454 controls (p = 0.008). In KCNQ2, a silent single nucleotide polymorphism (rs1801545) was found overrepresented in both epilepsy samples (IGE, p = 0.004). Conclusion: Sequence variations of the KCNQ2 and KCNQ3 genes may contribute to the etiology of common idiopathic epilepsy syndromes. GLOSSARY: BNFC = benign neonatal familial convulsions; bp = base pair; cRNA = complementary RNA; IAE = idiopathic absence epilepsy; IE = idiopathic epilepsy; IGE = idiopathic generalized epilepsy; JME = juvenile myoclonic epilepsy; OR = odds ratio; RE = rolandic epilepsy; SNP = single nucleotide polymorphism; WT = wild type.

DEPDC5 mutations in genetic focal epilepsies of childhood.

Recent studies reported DEPDC5 loss‐of‐function mutations in different focal epilepsy syndromes. Here we identified 1 predicted truncation and 2 missense mutations in 3 children with rolandic epilepsy (3 of 207). In addition, we identified 3 families with unclassified focal childhood epilepsies carrying predicted truncating DEPDC5 mutations (3 of 82). The detected variants were all novel, inherited, and present in all tested affected (n = 11) and in 7 unaffected family members, indicating low penetrance. Our findings extend the phenotypic spectrum associated with mutations in DEPDC5 and suggest that rolandic epilepsy, albeit rarely, and other nonlesional childhood epilepsies are among the associated syndromes. Ann Neurol 2014;75:788–792

Genetics of photosensitivity (photoparoxysmal response): a review.

Summary:  We present a review of phenotype–genotype correlation and the genetics of photosensitivity. The photoparoxysmal response in EEG (PPR) is still one of the best paradigms for exogenously triggered brain responses based on a genetic predisposition. The definition of the PPR phenotype requires multiple, precise methodologic guidelines. Individual factors such as age and gender but also other, unknown factors influence the expression of the PPR. For example, PPRs occur during adolescence and can disappear at a later age. As a consequence, it is difficult to assign nonaffected disease status correctly. Autosomal dominant inheritance has been found in clinical studies of relatives of PPR‐positive epilepsy and nonepilepsy subjects. Genetic heterogeneity of the PPR is obvious because the PPR also can be evoked in a number of autosomal recessive diseases. PPR is most commonly associated with idiopathic generalized epilepsies (IGEs) such as juvenile myoclonic epilepsy (JME). This comorbidity suggests that a genetic factor involved in photosensitivity also may influence the susceptibility for JME. Finding the gene for PPR also might represent a step forward in unraveling the genetic background of JME. The search for the genetic factors causing PPRs should focus on the genes affected in such epilepsies, such as genes (coding) for ion channels and neurotransmitters and their receptors. The expression of defined proteins with as‐yet‐undetermined functions, is changed in a few types of epilepsies with a mendelian mode of inheritance. These additional genes and the human equivalents of the genes found to be mutated in animal models also are candidates for molecular genetic studies of the PPR.

RBFOX1 and RBFOX3 Mutations in Rolandic Epilepsy

Partial deletions of the gene encoding the neuronal splicing regulator RBFOX1 have been reported in a range of neurodevelopmental diseases, including idiopathic generalized epilepsy. The RBFOX1 protein and its homologues (RBFOX2 and RBFOX3) regulate alternative splicing of many neuronal transcripts involved in the homeostatic control of neuronal excitability. In this study, we explored if structural microdeletions and exonic sequence variations in RBFOX1, RBFOX2, RBFOX3 confer susceptibility to rolandic epilepsy (RE), a common idiopathic focal childhood epilepsy. By high-density SNP array screening of 289 unrelated RE patients, we identified two hemizygous deletions, a 365 kb deletion affecting two untranslated 5′-terminal exons of RBFOX1 and a 43 kb deletion spanning exon 3 of RBFOX3. Exome sequencing of 242 RE patients revealed two novel probably deleterious variants in RBFOX1, a frameshift mutation (p.A233Vfs*74) and a hexanucleotide deletion (p.A299_A300del), and a novel nonsense mutation in RBFOX3 (p.Y287*). Although the three variants were inherited from unaffected parents, they were present in all family members exhibiting the RE trait clinically or electroencephalographically with only one exception. In contrast, no deleterious mutations of RBFOX1 and RBFOX3 were found in the exomes of 6503 non-RE subjects deposited in the Exome Variant Server database. The observed RBFOX3 exon 3 deletion and nonsense mutation suggest that RBFOX3 represents a novel risk factor for RE, indicating that exon deletions and truncating mutations of RBFOX1 and RBFOX3 contribute to the genetic variance of partial and generalized idiopathic epilepsy syndromes.

CDK6 associates with the centrosome during mitosis and is mutated in a large Pakistani family with primary microcephaly.

Autosomal recessive primary microcephaly (MCPH) is characterized by reduced head circumference, reduction in the size of the cerebral cortex with otherwise grossly normal brain structure and variable intellectual disability. MCPH is caused by mutations of 11 different genes which code for proteins implicated in cell division and cell cycle regulation. We studied a consanguineous eight-generation family from Pakistan with ten microcephalic children using homozygosity mapping and found a new MCPH locus at HSA 7q21.11-q21.3. Sanger sequencing of the most relevant candidate genes in this region revealed a homozygous single nucleotide substitution c.589G>A in CDK6, which encodes cyclin-dependent kinase 6. The mutation changes a highly conserved alanine at position 197 into threonine (p.Ala197Thr). Post hoc whole-exome sequencing corroborated this mutations identification as the causal variant. CDK6 is an important protein for the control of the cell cycle and differentiation of various cell types. We show here for the first time that CDK6 associates with the centrosome during mitosis; however, this was not observed in patient fibroblasts. Moreover, the mutant primary fibroblasts exhibited supernumerary centrosomes, disorganized microtubules and mitotic spindles, an increased centrosome nucleus distance, reduced cell proliferation and impaired cell motility and polarity. Upon ectopic expression of the mutant protein and knockdown of CDK6 through shRNA, we noted similar effects. We propose that the identified CDK6 mutation leads to reduced cell proliferation and impairs the correct functioning of the centrosome in microtubule organization and its positioning near the nucleus which are key determinants during neurogenesis.

Burden Analysis of Rare Microdeletions Suggests a Strong Impact of Neurodevelopmental Genes in Genetic Generalised Epilepsies

Genetic generalised epilepsy (GGE) is the most common form of genetic epilepsy, accounting for 20% of all epilepsies. Genomic copy number variations (CNVs) constitute important genetic risk factors of common GGE syndromes. In our present genome-wide burden analysis, large (≥ 400 kb) and rare (< 1%) autosomal microdeletions with high calling confidence (≥ 200 markers) were assessed by the Affymetrix SNP 6.0 array in European case-control cohorts of 1,366 GGE patients and 5,234 ancestry-matched controls. We aimed to: 1) assess the microdeletion burden in common GGE syndromes, 2) estimate the relative contribution of recurrent microdeletions at genomic rearrangement hotspots and non-recurrent microdeletions, and 3) identify potential candidate genes for GGE. We found a significant excess of microdeletions in 7.3% of GGE patients compared to 4.0% in controls (P = 1.8 x 10-7; OR = 1.9). Recurrent microdeletions at seven known genomic hotspots accounted for 36.9% of all microdeletions identified in the GGE cohort and showed a 7.5-fold increased burden (P = 2.6 x 10-17) relative to controls. Microdeletions affecting either a gene previously implicated in neurodevelopmental disorders (P = 8.0 x 10-18, OR = 4.6) or an evolutionarily conserved brain-expressed gene related to autism spectrum disorder (P = 1.3 x 10-12, OR = 4.1) were significantly enriched in the GGE patients. Microdeletions found only in GGE patients harboured a high proportion of genes previously associated with epilepsy and neuropsychiatric disorders (NRXN1, RBFOX1, PCDH7, KCNA2, EPM2A, RORB, PLCB1). Our results demonstrate that the significantly increased burden of large and rare microdeletions in GGE patients is largely confined to recurrent hotspot microdeletions and microdeletions affecting neurodevelopmental genes, suggesting a strong impact of fundamental neurodevelopmental processes in the pathogenesis of common GGE syndromes.