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.

Exonic microdeletions of the gephyrin gene impair GABAergic synaptic inhibition in patients with idiopathic generalized epilepsy.

Gephyrin is a postsynaptic scaffolding protein, essential for the clustering of glycine and γ-aminobutyric acid type-A receptors (GABAARs) at inhibitory synapses. An impairment of GABAergic synaptic inhibition represents a key pathway of epileptogenesis. Recently, exonic microdeletions in the gephyrin (GPHN) gene have been associated with neurodevelopmental disorders including autism spectrum disorder, schizophrenia and epileptic seizures. Here we report the identification of novel exonic GPHN microdeletions in two patients with idiopathic generalized epilepsy (IGE), representing the most common group of genetically determined epilepsies. The identified GPHN microdeletions involve exons 5-9 (Δ5-9) and 2-3 (Δ2-3), both affecting the gephyrin G-domain. Molecular characterization of the GPHN Δ5-9 variant demonstrated that it perturbs the clustering of regular gephyrin at inhibitory synapses in cultured mouse hippocampal neurons in a dominant-negative manner, resulting in a significant loss of γ2-subunit containing GABAARs. GPHN Δ2-3 causes a frameshift resulting in a premature stop codon (p.V22Gfs*7) leading to haplo-insufficiency of the gene. Our results demonstrate that structural exonic microdeletions affecting the GPHN gene constitute a rare genetic risk factor for IGE and other neuropsychiatric disorders by an impairment of the GABAergic inhibitory synaptic transmission.

A common SCN1A splice-site polymorphism modifies the effect of carbamazepine on cortical excitability--a pharmacogenetic transcranial magnetic stimulation study.

OBJECTIVE: SCN1A encodes the alpha subunit of the voltage-gated sodium channel and plays a crucial role in several epilepsy syndromes. The common SCN1A splice-site polymorphism rs3812718 (IVS5N+5 G>A) might contribute to the pathophysiology underlying genetic generalized epilepsies and is associated with electrophysiologic properties of the channel and the effect of sodium-channel blocking antiepileptic drugs. We assessed the effects of the rs3812718 genotype on cortical excitability at baseline and after administration of carbamazepine in order to investigate the mechanism of this association. METHODS: Paired-pulse transcranial magnetic stimulation (TMS) was applied in 92 healthy volunteers with the homozygous genotypes AA or GG of rs3812718 at baseline and after application of 400 mg of carbamazepine or placebo in a double-blind, randomized, crossover design. Resting motor threshold (RMT), short interval intracortical inhibition (SICI), intracortical facilitation (ICF), and cortical silent period (CSP) were determined. RESULTS: At baseline there was no significant difference in any TMS parameter. Genotype GG was associated with a higher carbamazepine-induced increase in CSP duration as compared to AA (multivariate analysis of covariance [MANCOVA], p = 0.013). An expected significant increase in RMT was genotype independent. SIGNIFICANCE: We found that the rs3812718 genotype modifies the effect of carbamazepine on CSP duration (mainly reflecting modulation of γ-aminobutyric acid (GABA)ergic inhibition), but not on RMT (mainly reflecting modulation of voltage-gated sodium channels). This provides evidence that rs3812718 affects the pharmacoresponse to carbamazepine via an effect on GABAergic cortical interneurons. Our results also confirm that TMS is useful to investigate the effect of genetic variants on cortical excitability and pharmacoresponse.

Copy number variants are frequent in genetic generalized epilepsy with intellectual disability.

OBJECTIVE: We examined whether copy number variants (CNVs) were more common in those with a combination of intellectual disability (ID) and genetic generalized epilepsy (GGE) than in those with either phenotype alone via a case-control study. METHODS: CNVs contribute to the genetics of multiple neurodevelopmental disorders with complex inheritance, including GGE and ID. Three hundred fifty-nine probands with GGE and 60 probands with ID-GGE were screened for GGE-associated recurrent microdeletions at 15q13.3, 15q11.2, and 16p13.11 via quantitative PCR or loss of heterozygosity. Deletions were confirmed by comparative genomic hybridization (CGH). ID-GGE probands also had genome-wide CGH. RESULTS: ID-GGE probands showed a significantly higher rate of CNVs compared with probands with GGE alone, with 17 of 60 (28%) ID-GGE probands having one or more potentially causative CNVs. The patients with ID-GGE had a 3-fold-higher rate of the 3 GGE-associated recurrent microdeletions than probands with GGE alone (10% vs 3%, p = 0.02). They also showed a high rate (13/60, 22%) of rare CNVs identified using genome-wide CGH. CONCLUSIONS: This study shows that CNVs are common in those with ID-GGE with recurrent deletions at 15q13.3, 15q11.2, and 16p13.11, particularly enriched compared with individuals with GGE or ID alone. Recurrent CNVs are likely to act as risk factors for multiple phenotypes not just at the population level, but also in any given individual. Testing for CNVs in ID-GGE will have a high diagnostic yield in a clinical setting and will inform genetic counseling.

EXOSC3 mutations in isolated cerebellar hypoplasia and spinal anterior horn involvement.

Pontocerebellar hypoplasia (PCH) type 1 is characterized by the co-occurrence of spinal anterior horn involvement and hypoplasia of the cerebellum and pons. EXOSC3 has been recently defined as a major cause of PCH type 1. Three different phenotypes showing variable severity have been reported. We identified a homozygous mutation [c.395A > C/p.D132A] in EXOSC3 in four patients with muscle hypotonia, developmental delay, spinal anterior horn involvement, and prolonged survival, consistent with the "mild PCH1 phenotype". Interestingly, isolated cerebellar hypoplasia limited to the hemispheres or involving both hemispheres and vermis was the main neuroradiologic finding, whereas the pontine volume was in the normal range for age. These findings strongly suggest that analysis of the EXOSC3 gene should be recommended also in patients with spinal anterior horn involvement and isolated cerebellar hypoplasia.

Rare exonic deletions of the RBFOX1 gene increase risk of idiopathic generalized epilepsy.

PURPOSE: Structural variations disrupting the gene encoding the neuron-specific splicing regulator RBFOX1 have been reported in three patients exhibiting epilepsy in comorbidity with other neuropsychiatric disorders. Consistently, the Rbfox1 knockout mouse model showed an increased susceptibility of seizures. The present candidate gene study tested whether exon-disrupting deletions of RBFOX1 increase the risk of idiopathic generalized epilepsies (IGEs), representing the largest group of genetically determined epilepsies. METHODS: Screening of microdeletions (size: >40 kb, coverage >20 markers) affecting the genomic sequence of the RBFOX1 gene was carried out by high-resolution single-nucleotide polymorphism (SNP) arrays in 1,408 European patients with idiopathic generalized epilepsy (IGE) and 2,256 population controls. Validation of RBFOX1 deletions and familial segregation analysis were performed by quantitative polymerase chain reaction (qPCR). KEY FINDINGS: We detected five exon-disrupting RBFOX1 deletions in the IGE patients, whereas none was observed in the controls (p = 0.008, Fisher's exact test). The size of the exonic deletions ranged from 68 to 896 kb and affected the untranslated 5'-terminal RBFOX1 exons. Segregation analysis in four families indicated that the deletions were inherited, display incomplete penetrance, and heterogeneous cosegregation patterns with IGE. SIGNIFICANCE: Rare deletions affecting the untranslated 5'-terminal RBFOX1 exons increase risk of common IGE syndromes. Variable expressivity, incomplete penetrance, and heterogeneous cosegregation patterns suggest that RBFOX1 deletions act as susceptibility factor in a genetically complex etiology, where heterogeneous combinations of genetic factors determine the disease phenotype.

Exon-disrupting deletions of NRXN1 in idiopathic generalized epilepsy.

PURPOSE: Neurexins are neuronal adhesion molecules located in the presynaptic terminal, where they interact with postsynaptic neuroligins to form a transsynaptic complex required for efficient neurotransmission in the brain. Recently, deletions and point mutations of the neurexin 1 (NRXN1) gene have been associated with a broad spectrum of neuropsychiatric disorders. This study aimed to investigate if NRXN1 deletions also increase the risk of idiopathic generalized epilepsies (IGEs). METHODS: We screened for deletions involving the NRXN1 gene in 1,569 patients with IGE and 6,201 controls using high-density oligonucleotide microarrays. KEY FINDINGS: We identified exon-disrupting deletions of NRXN1 in 5 of 1,569 patients with IGE and 2 of 6,201 control individuals (p = 0.0049; odds ratio (OR) 9.91, 95% confidence interval (CI) 1.92-51.12). A complex familial segregation pattern in the IGE families was observed, suggesting that heterozygous NRXN1 deletions are susceptibility variants. Intriguingly, we identified a second large copy number variant in three of five index patients, supporting an involvement of heterogeneous susceptibility alleles in the etiology of IGE. SIGNIFICANCE: We conclude that exon-disrupting deletions of NRXN1 represent a genetic risk factor in the genetically complex predisposition of common IGE syndromes.

Mutations in FKBP10 can cause a severe form of isolated Osteogenesis imperfecta.

BACKGROUND: Mutations in the FKBP10 gene were first described in patients with Osteogenesis imperfecta type III. Two follow up reports found FKBP10 mutations to be associated with Bruck syndrome type 1, a rare disorder characterized by congenital contractures and bone fragility. This raised the question if the patients in the first report indeed had isolated Osteogenesis imperfecta or if Bruck syndrome would have been the better diagnosis. METHODS: The patients described here are affected by severe autosomal recessive Osteogenesis imperfecta without contractures. RESULTS: Homozygosity mapping identified FKBP10 as a candidate gene, and sequencing revealed a base pair exchange that causes a C-terminal premature stop codon in this gene. CONCLUSIONS: Our study demonstrates that FKBP10 mutations not only cause Bruck syndrome or Osteogenesis imperfecta type III but can result in a severe type of isolated Osteogenesis imperfecta type IV with prenatal onset. Furthermore, it adds dentinogenesis imperfecta to the spectrum of clinical symptoms associated with FKBP10 mutations.

Recurrent microdeletions at 15q11.2 and 16p13.11 predispose to idiopathic generalized epilepsies.

Idiopathic generalized epilepsies account for 30% of all epilepsies. Despite a predominant genetic aetiology, the genetic factors predisposing to idiopathic generalized epilepsies remain elusive. Studies of structural genomic variations have revealed a significant excess of recurrent microdeletions at 1q21.1, 15q11.2, 15q13.3, 16p11.2, 16p13.11 and 22q11.2 in various neuropsychiatric disorders including autism, intellectual disability and schizophrenia. Microdeletions at 15q13.3 have recently been shown to constitute a strong genetic risk factor for common idiopathic generalized epilepsy syndromes, implicating that other recurrent microdeletions may also be involved in epileptogenesis. This study aimed to investigate the impact of five microdeletions at the genomic hotspot regions 1q21.1, 15q11.2, 16p11.2, 16p13.11 and 22q11.2 on the genetic risk to common idiopathic generalized epilepsy syndromes. The candidate microdeletions were assessed by high-density single nucleotide polymorphism arrays in 1234 patients with idiopathic generalized epilepsy from North-western Europe and 3022 controls from the German population. Microdeletions were validated by quantitative polymerase chain reaction and their breakpoints refined by array comparative genomic hybridization. In total, 22 patients with idiopathic generalized epilepsy (1.8%) carried one of the five novel microdeletions compared with nine controls (0.3%) (odds ratio = 6.1; 95% confidence interval 2.8-13.2; chi(2) = 26.7; 1 degree of freedom; P = 2.4 x 10(-7)). Microdeletions were observed at 1q21.1 [Idiopathic generalized epilepsy (IGE)/control: 1/1], 15q11.2 (IGE/control: 12/6), 16p11.2 IGE/control: 1/0, 16p13.11 (IGE/control: 6/2) and 22q11.2 (IGE/control: 2/0). Significant associations with IGEs were found for the microdeletions at 15q11.2 (odds ratio = 4.9; 95% confidence interval 1.8-13.2; P = 4.2 x 10(-4)) and 16p13.11 (odds ratio = 7.4; 95% confidence interval 1.3-74.7; P = 0.009). Including nine patients with idiopathic generalized epilepsy in this cohort with known 15q13.3 microdeletions (IGE/control: 9/0), parental transmission could be examined in 14 families. While 10 microdeletions were inherited (seven maternal and three paternal transmissions), four microdeletions occurred de novo at 15q13.3 (n = 1), 16p13.11 (n = 2) and 22q11.2 (n = 1). Eight of the transmitting parents were clinically unaffected, suggesting that the microdeletion itself is not sufficient to cause the epilepsy phenotype. Although the microdeletions investigated are individually rare (<1%) in patients with idiopathic generalized epilepsy, they collectively seem to account for a significant fraction of the genetic variance in common idiopathic generalized epilepsy syndromes. The present results indicate an involvement of microdeletions at 15q11.2 and 16p13.11 in epileptogenesis and strengthen the evidence that recurrent microdeletions at 15q11.2, 15q13.3 and 16p13.11 confer a pleiotropic susceptibility effect to a broad range of neuropsychiatric disorders.

Familial and sporadic 15q13.3 microdeletions in idiopathic generalized epilepsy: precedent for disorders with complex inheritance.

Microdeletion at chromosomal position 15q13.3 has been described in intellectual disability, autism spectrum disorders, schizophrenia and recently in idiopathic generalized epilepsy (IGE). Using independent IGE cohorts, we first aimed to confirm the association of 15q13.3 deletions and IGE. We then set out to determine the relative occurrence of sporadic and familial cases and to examine the likelihood of having seizures for individuals with the microdeletion in familial cases. The 15q13.3 microdeletion was identified in 7 of 539 (1.3%) unrelated cases of IGE using quantitative PCR or SNP arrays and confirmed by array comparative genomic hybridization analysis using probes specific to the 15q13.3 region. The inheritance of this lesion was tracked using family studies. Of the seven microdeletions identified in probands, three were de novo, two were transmitted from an unaffected parent and in two cases the parents were unavailable. Non-penetrance of the microdeletion was identified in 4/7 pedigrees and three pedigrees included other family members with IGE who lacked the 15q13.3 deletion. The odds ratio is 68 (95% confidence interval 29-181), indicating a pathogenic lesion predisposing to epilepsy with complex inheritance and incomplete penetrance for the IGE component of the phenotype in multiplex families.