[Show abstract][Hide abstract] ABSTRACT: The epileptic encephalopathies are a clinically and aetiologically heterogeneous subgroup of epilepsy syndromes. Most epileptic encephalopathies have a genetic cause and patients are often found to carry a heterozygous de novo mutation in one of the genes associated with the disease entity. Occasionally recessive mutations are identified: a recent publication described a distinct neonatal epileptic encephalopathy (MIM 615905) caused by autosomal recessive mutations in the SLC13A5 gene. Here, we report eight additional patients belonging to four different families with autosomal recessive mutations in SLC13A5. SLC13A5 encodes a high affinity sodium-dependent citrate transporter, which is expressed in the brain. Neurons are considered incapable of de novo synthesis of tricarboxylic acid cycle intermediates; therefore they rely on the uptake of intermediates, such as citrate, to maintain their energy status and neurotransmitter production. The effect of all seven identified mutations (two premature stops and five amino acid substitutions) was studied in vitro, using immunocytochemistry, selective western blot and mass spectrometry. We hereby demonstrate that cells expressing mutant sodium-dependent citrate transporter have a complete loss of citrate uptake due to various cellular loss-of-function mechanisms. In addition, we provide independent proof of the involvement of autosomal recessive SLC13A5 mutations in the development of neonatal epileptic encephalopathies, and highlight teeth hypoplasia as a possible indicator for SLC13A5 screening. All three patients who tried the ketogenic diet responded well to this treatment, and future studies will allow us to ascertain whether this is a recurrent feature in this severe disorder.
[Show abstract][Hide abstract] ABSTRACT: Idiopathic/genetic generalized epilepsy (IGE/GGE) is characterized by seizures, which start and rapidly engage widely distributed networks, and result in symptoms such as absences, generalized myoclonic and primary generalized tonic-clonic seizures. Although routine magnetic resonance imaging is apparently normal, many studies have reported structural alterations in IGE/GGE patients using diffusion tensor imaging and voxel-based morphometry. Changes have also been reported in functional networks during generalized spike wave discharges. However, network function in the resting-state without epileptiforme discharges has been less well studied. We hypothesize that resting-state networks are more representative of the underlying pathophysiology and abnormal network synchrony. We studied functional network connectivity derived from whole-brain magnetoencephalography recordings in thirteen IGE/GGE and nineteen healthy controls. Using graph theoretical network analysis, we found a widespread increase in connectivity in patients compared to controls. These changes were most pronounced in the motor network, the mesio-frontal and temporal cortex. We did not, however, find any significant difference between the normalized clustering coefficients, indicating preserved gross network architecture. Our findings suggest that increased resting state connectivity could be an important factor for seizure spread and/or generation in IGE/GGE, and could serve as a biomarker for the disease.
PLoS ONE 09/2015; 10(9):e0138119. DOI:10.1371/journal.pone.0138119 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: ATP-binding cassette transporter B1 (ABCB1; P-glycoprotein; multidrug resistance protein 1) is an adenosine triphosphate (ATP)-dependent efflux transporter located in the plasma membrane of many different cell types. Numerous structurally unrelated compounds, including drugs and environmental toxins, have been identified as substrates. ABCB1 limits the absorption of xenobiotics from the gut lumen, protects sensitive tissues (e.g. the brain, fetus and testes) from xenobiotics and is involved in biliary and renal secretion of its substrates. In recent years, a large number of polymorphisms of the ABCB1 [ATP-binding cassette, sub-family B (MDR/TAP), member 1] gene have been described. The variants 1236C>T (rs1128503, p.G412G), 2677G>T/A (rs2032582, p.A893S/T) and 3435C>T (rs1045642, p.I1145I) occur at high allele frequencies and create a common haplotype; therefore, they have been most widely studied. This review provides an overview of clinical studies published between 2002 and March 2015. In summary, the effect of ABCB1 variation on P-glycoprotein expression (messenger RNA and protein expression) and/or activity in various tissues (e.g. the liver, gut and heart) appears to be small. Although polymorphisms and haplotypes of ABCB1 have been associated with alterations in drug disposition and drug response, including adverse events with various ABCB1 substrates in different ethnic populations, the results have been majorly conflicting, with limited clinical relevance. Future research activities are warranted, considering a deep-sequencing approach, as well as well-designed clinical studies with appropriate sample sizes to elucidate the impact of rare ABCB1 variants and their potential consequences for effect sizes.
[Show abstract][Hide abstract] ABSTRACT: Restless legs syndrome (RLS) is characterized by unpleasant sensations in the legs and an uncontrollable urge to move them in order to gain relief. Higher frequencies of RLS have been reported in systemic lupus, multiple sclerosis, rheumatoid arthritis and atopic dermatitis.
Since the disease-related stress present in psoriasis is similar to the stress of those diseases, we aimed to study the frequency of RLS in a German cohort of patients with psoriasis.
300 patients with psoriasis and 300 healthy controls were evaluated for RLS symptoms in this study.
While 17% (n = 51) of patients with psoriasis reported symptoms of RLS, only 4% (n = 12) of individuals without psoriasis suffered from RLS symptoms (95% confidence interval: 0.08 - 0.18, p<0.01). In patients with psoriasis and RLS the average RLS score was 16.0 ± 9.2 whereas individuals with RLS in the control group had an average RLS score of 13.5 ± 7.1.
Our findings indicate an increased frequency of RLS in patients with psoriasis, suggesting screening patients with psoriasis for the presence of RLS as a well-treatable co-morbidity.
[Show abstract][Hide abstract] ABSTRACT: Epileptic encephalopathies are a phenotypically and genetically heterogeneous group of severe epilepsies accompanied by intellectual disability and other neurodevelopmental features –6. Using next-generation sequencing, we identified four different de novo mutations in KCNA2, encoding the potassium channel K V .2, in six isolated patients with epileptic encephalopathy (one mutation recurred three times independently). Four individuals presented with febrile and multiple afebrile, often focal seizure types, multifocal epileptiform discharges strongly activated by sleep, mild to moderate intellectual disability, delayed speech development and sometimes ataxia. Functional studies of the two mutations associated with this phenotype showed almost complete loss of function with a dominant-negative effect. Two further individuals presented with a different and more severe epileptic encephalopathy phenotype. They carried mutations inducing a drastic gain-of-function effect leading to permanently open channels. These results establish KCNA2 as a new gene involved in human neurodevelopmental disorders through two different mechanisms, predicting either hyperexcitability or electrical silencing of K V .2-expressing neurons. Many of the voltage-gated potassium channels (K V 1–K V 12) are expressed in the central nervous system (CNS), having an important role in neuronal excitability and neurotransmitter release 7. Mutations in potassium channel–encoding genes cause different neurological diseases, including benign familial neonatal seizures (KCNQ2, encoding K V 7.2; KCNQ3, encoding K V 7.3) 8–10 , neonatal epileptic encephalopathy (KCNQ2) 11,12 , episodic ataxia type 1 (EA1) (KCNA1, encoding K V 1.1) 13 and peripheral nerve hyperexcitability (KCNA1, KCNQ2) 13–15. In addition, antibodies against K V 1.1 or associated proteins such as contactin-associated protein 2 (CASPR2) or leucine-rich, glioma-inactivated 1 (LGI1) cause limbic encephalitis or neuromyotonia 16. Therefore, potassium channel genes represent interesting candidates for neurodevelopmental disorders. To identify mutations in presumed genetic forms of epilepsy, we designed a targeted resequencing panel 17 comprising 265 known and 220 candidate genes for epilepsy (Supplementary Table 1). Screening a pilot cohort of 33 patients, we identified mutations in known epilepsy-related genes in 16 cases 17. We evaluated the remaining 17 cases for mutations in candidate genes (Supplementary Note), which led to the detection of a heterozygous de novo mutation in KCNA2, c.1214C>T (encoding p.Pro405Leu), affecting the highly conserved pore domain of the voltage-gated potassium channel K V 1.2 (NM_004974, CCDS827). This mutation was not present in control databases (1000 Genomes Project, Exome Variant Server (EVS), dbSNP138 or the Exome Aggregation Consortium (ExAC) database). The affected female (patient 1) carrying this mutation had unre-markable early development until the onset of epilepsy at 17 months of age. The phenotype included febrile and afebrile alternating hemiclonic seizures and status epilepticus, reminiscent of Dravet syndrome. The electroencephalogram (EEG) showed multifocal spikes with marked activation during sleep. After seizure onset, ataxia and delay of psychomotor and language development became apparent. She had postnatal short stature, growth hormone deficiency and hypothyroidism. Seizures and ataxia responded poorly to antiepileptic drugs (topiramate, oxcarbazepine, valproic acid and bromide), including acetazolamide (known to be effective in EA1 caused by mutations in KCNA1; ref. 18). At last follow-up at 8 years of age, she had remained seizure free for the past 6 months without previous change of medication. We identified further KCNA2 mutations in several parallel studies (Supplementary Fig. 1). First, we performed whole-exome sequenc-ing in 86 parent-offspring trios with epileptic encephalopathy (31 with Dravet syndrome negative for mutations in SCN1A, 39 with myoclonic-atonic epilepsy (MAE) and 16 with electrical status epi-lepticus in slow-wave sleep (ESES)). Second, we performed panel sequencing (Supplementary Note) in 147 adults with a broad spectrum of epilepsy phenotypes associated with intellectual disability. Third, we performed whole-exome sequencing in an adult cohort of 10 independent trios with severe epilepsy and intellectual disability and whole-exome sequencing in another cohort of 12 independent, isolated index cases with early-onset ataxia and epilepsy. We identified six additional independent cases with previously unre-ported heterozygous KCNA2 variants (Table 1, Supplementary Fig. 2 and Supplementary Note). Patient 2 (initially classified as having MAE) carried the de novo mutation c.788T>C (encoding p.Ile263Thr). Patient 3 (intellectual disability with neonatal-onset focal epilepsy and cerebel-lar hypoplasia) carried the variant c.440G>A (encoding p.Arg147Lys), of unknown inheritance. We considered p.Arg147Lys to be a variant of unknown relevance because (i) it could not be confirmed as de novo, (ii) it was predicted to be benign using seven of nine prediction tools, (iii) a lysine occurs naturally at this position in Drosophila melanogaster and zebrafish, and (iv) the change did not show functional consequences (Supplementary Fig. 3, Supplementary Tables 1 and 2, and Supplementary Note). Patient 4 (initially classified as having Dravet
[Show abstract][Hide abstract] ABSTRACT: Epileptic encephalopathies are a phenotypically and genetically heterogeneous group of severe epilepsies accompanied by intellectual disability and other neurodevelopmental features. Using next-generation sequencing, we identified four different de novo mutations in KCNA2, encoding the potassium channel KV1.2, in six isolated patients with epileptic encephalopathy (one mutation recurred three times independently). Four individuals presented with febrile and multiple afebrile, often focal seizure types, multifocal epileptiform discharges strongly activated by sleep, mild to moderate intellectual disability, delayed speech development and sometimes ataxia. Functional studies of the two mutations associated with this phenotype showed almost complete loss of function with a dominant-negative effect. Two further individuals presented with a different and more severe epileptic encephalopathy phenotype. They carried mutations inducing a drastic gain-of-function effect leading to permanently open channels. These results establish KCNA2 as a new gene involved in human neurodevelopmental disorders through two different mechanisms, predicting either hyperexcitability or electrical silencing of KV1.2-expressing neurons.
[Show abstract][Hide abstract] ABSTRACT: Objective: To test whether mutations in gamma-aminobutyric acid type A receptor (GABAA-R) subunit genes contribute to the etiology of Rolandic epilepsy (RE) or its atypical variants (ARE).
Methods: We performed exome sequencing to compare the frequency of variants in 18 GABAA-R genes in 204 European patients with RE/ARE versus 728 platform matched controls. Identified GABRG2 variants were functionally assessed for protein stability, trafficking, postsynaptic clustering and receptor function.
Results: Out of 18 screened GABAA-R genes, we detected an enrichment of rare variants in the GABRG2 gene in RE/ARE patients (5/204, 2.45%) in comparison to controls (1/723, 0.14%) (OR = 18.07, 95% CI = 2.01 – 855.07, p = 0.0024, pcorr = 0.043). We identified a GABRG2 splice variant (c.549-3T>G) in two unrelated patients as well as three nonsynonymous variations in this gene (p.G257R, p.R323Q, p.I389V). Functional assessment showed reduced surface expression of p.G257R and decreased GABA-evoked currents for p.R323Q. The p.G257R mutation displayed diminished levels of palmitoylation, a posttranslational modification crucial for trafficking of proteins to the cell membrane. Enzymatically raised palmitoylation levels restored the surface expression of the p.G257R variant γ2-subunit.
Interpretation: The statistical association and the functional evidence suggest that mutations of the GABRG2 gene may increase the risk of RE/ARE. Restoring the impaired membrane trafficking of some GABRG2 mutations by enhancing palmitoylation might be an interesting therapeutic approach to reverse the pathogenic effect of such mutants. This article is protected by copyright. All rights reserved.
Annals of Neurology 02/2015; 77(6). DOI:10.1002/ana.24395 · 9.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Progressive myoclonus epilepsies (PMEs) are a group of rare, inherited disorders manifesting with action myoclonus, tonic-clonic seizures and ataxia. We sequenced the exomes of 84 unrelated individuals with PME of unknown cause and molecularly solved 26 cases (31%). Remarkably, a recurrent de novo mutation, c.959G>A (p.Arg320His), in KCNC1 was identified as a new major cause for PME. Eleven unrelated exome-sequenced (13%) and two affected individuals in a secondary cohort (7%) had this mutation. KCNC1 encodes KV3.1, a subunit of the KV3 voltage-gated potassium ion channels, which are major determinants of high-frequency neuronal firing. Functional analysis of the Arg320His mutant channel showed a dominant-negative loss-of-function effect. Ten cases had pathogenic mutations in known PME-associated genes (NEU1, NHLRC1, AFG3L2, EPM2A, CLN6 and SERPINI1). Identification of mutations in PRNP, SACS and TBC1D24 expand their phenotypic spectra to PME. These findings provide insights into the molecular genetic basis of PME and show the role of de novo mutations in this disease entity.
[Show abstract][Hide abstract] ABSTRACT: Mutations in SCN1A and other ion channel genes can cause different epileptic phenotypes, but the precise mechanisms underlying the development of hyperexcitable networks are largely unknown. Here, we present a multisystem analysis of an SCN1A mouse model carrying the NaV1.1-R1648H mutation, which causes febrile seizures and epilepsy in humans. We found a ubiquitous hypoexcitability of interneurons in thalamus, cortex, and hippocampus, without detectable changes in excitatory neurons. Interestingly, somatic Na(+) channels in interneurons and persistent Na(+) currents were not significantly changed. Instead, the key mechanism of interneuron dysfunction was a deficit of action potential initiation at the axon initial segment that was identified by analyzing action potential firing. This deficit increased with the duration of firing periods, suggesting that increased slow inactivation, as recorded for recombinant mutated channels, could play an important role. The deficit in interneuron firing caused reduced action potential-driven inhibition of excitatory neurons as revealed by less frequent spontaneous but not miniature IPSCs. Multiple approaches indicated increased spontaneous thalamocortical and hippocampal network activity in mutant mice, as follows: (1) more synchronous and higher-frequency firing was recorded in primary neuronal cultures plated on multielectrode arrays; (2) thalamocortical slices examined by field potential recordings revealed spontaneous activities and pathological high-frequency oscillations; and (3) multineuron Ca(2+) imaging in hippocampal slices showed increased spontaneous neuronal activity. Thus, an interneuron-specific generalized defect in action potential initiation causes multisystem disinhibition and network hyperexcitability, which can well explain the occurrence of seizures in the studied mouse model and in patients carrying this mutation.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 11/2014; 34(45):14874-89. DOI:10.1523/JNEUROSCI.0721-14.2014 · 6.34 Impact Factor