Increased frequency of proinflammatory CD4 T cells and pathological levels of serum neurofilament light chain in adult drug-resistant epilepsy.

OBJECTIVE: Adult drug-resistant epilepsy (DRE) is associated with significant morbidity. Infiltration of immune cells is observed in DRE epileptic foci; however, the relation between DRE and the peripheral immune cell compartment remains only partially understood. We aimed to investigate differences in immune cell populations, cytokines, and neurodegenerative biomarkers in the peripheral blood of subjects with epilepsy versus healthy controls, and in DRE compared to well-controlled epilepsy (WCE). METHODS: Peripheral blood mononuclear cells and serum from >120 age- and sex-matched adults suffering from focal onset epilepsy and controls were analyzed by multipanel flow cytometry, multiplex immunoassays, and ultrasensitive single molecule array. RESULTS: Using a data-driven analytical approach, we identified that CD4 T cells in the peripheral blood are present in a higher proportion in DRE patients. Moreover, we observed that the frequency of CD4 T cells expressing proinflammatory cytokines interleukin (IL)-17A, IL-22, tumor necrosis factor, interferon-γ, and granulocyte-macrophage colony-stimulating factor, but not anti-inflammatory cytokines IL-10 and IL-4, is elevated in the peripheral blood of DRE subjects compared to WCE. In parallel, we found that Th17-related circulating proinflammatory cytokines are elevated, but Th2-related cytokine IL-4 is reduced, in the serum of epilepsy and DRE subjects. As Th17 cells can exert neurotoxicity, we measured levels of serum neurofilament light chain (sNfL), a marker of neuronal injury. We found significantly elevated levels of sNfL in DRE compared to controls, especially among older individuals. SIGNIFICANCE: Our data support that DRE is associated with an expansion of the CD4 Tcell subset in the peripheral blood and with a shift toward a proinflammatory Th17/Th1 CD4 Tcell immune profile. Our results further show that pathological levels of sNfL are more frequent in DRE, supporting a potential neurodegenerative component in adult DRE. With this work, we provide evidence for novel potential inflammatory and degenerative biomarkers in DRE.

Variants in CHRNB2 and CHRNA4 Identified in Patients with Insular Epilepsy.

PURPOSE: Our purpose was to determine the role of CHRNA4 and CHRNB2 in insular epilepsy. METHOD: We identified two patients with drug-resistant predominantly sleep-related hypermotor seizures, one harboring a heterozygous missense variant (c.77C>T; p. Thr26Met) in the CHRNB2 gene and the other a heterozygous missense variant (c.1079G>A; p. Arg360Gln) in the CHRNA4 gene. The patients underwent electrophysiological and neuroimaging studies, and we performed functional characterization of the p. Thr26Met (c.77C>T) in the CHRNB2 gene. RESULTS: We localized the epileptic foci to the left insula in the first case (now seizure-free following epilepsy surgery) and to both insulae in the second case. Based on tools predicting the possible impact of amino acid substitutions on the structure and function of proteins (sorting intolerant from tolerant and PolyPhen-2), variants identified in this report could be deleterious. Functional expression in human cell lines of α4ß2 (wild-type), α4ß2-Thr26Met (homozygote), and α4ß2/ß2-Thr26Met (heterozygote) nicotinic acetylcholine receptors revealed that the mutant subunit led to significantly higher whole-cell nicotinic currents. This feature was observed in both homo- and heterozygous conditions and was not accompanied by major alterations of the current reversal potential or the shape of the concentration-response relation. CONCLUSIONS: This study suggests that variants in CHRNB2 and CHRNA4, initially linked to autosomal dominant nocturnal frontal lobe epilepsy, are also found in patients with predominantly sleep-related insular epilepsy. Although the reported variants should be considered of unknown clinical significance for the moment, identification of additional similar cases and further functional studies could eventually strengthen this association.

Utility of magnetic source imaging in nonlesional focal epilepsy: a prospective study.

OBJECTIVE: For patients with nonlesional refractory focal epilepsy (NLRFE), localization of the epileptogenic zone may be more arduous than for other types of epilepsy and frequently requires information from multiple noninvasive presurgical modalities and intracranial EEG (icEEG). In this prospective, blinded study, the authors assessed the clinical added value of magnetic source imaging (MSI) in the presurgical evaluation of patients with NLRFE. METHODS: This study prospectively included 57 consecutive patients with NLRFE who were considered for epilepsy surgery. All patients underwent noninvasive presurgical evaluation and then MSI. To determine the surgical plan, discussion of the results of the presurgical evaluation was first undertaken while discussion participants were blinded to the MSI results. MSI results were then presented. MSI influence on the initial management plan was assessed. RESULTS: MSI results influenced patient management in 32 patients. MSI results led to the following changes in surgical strategy in 14 patients (25%): allowing direct surgery in 6 patients through facilitating the detection of subtle cortical dysplasia in 4 patients and providing additional concordant diagnostic information to other presurgical workup in another 2 patients; rejection of surgery in 3 patients originally deemed surgical candidates; change of plan from direct surgery to icEEG in 2 patients; and allowing icEEG in 3 patients deemed not surgical candidates. MSI results led to changed electrode locations and contact numbers in another 18 patients. Epilepsy surgery was performed in 26 patients influenced by MSI results and good surgical outcome was achieved in 21 patients. CONCLUSIONS: This prospective, blinded study showed that information provided by MSI allows more informed icEEG planning and surgical outcome in a significant percentage of patients with NLRFE and should be included in the presurgical workup in those patients.

Autosomal-dominant adult neuronal ceroid lipofuscinosis caused by duplication in DNAJC5 initially missed by Sanger and whole-exome sequencing.

Adult-onset neuronal ceroid lipofuscinoses (ANCL, Kufs disease) are rare hereditary neuropsychiatric disorders characterized by intralysosomal accumulation of ceroid in tissues. The ceroid accumulation primarily affects the brain, leading to neuronal loss and progressive neurodegeneration. Although several causative genes have been identified (DNAJC5, CLN6, CTSF, GRN, CLN1, CLN5, ATP13A2), the genetic underpinnings of ANCL in some families remain unknown. Here we report one family with autosomal dominant (AD) Kufs disease caused by a 30 bp in-frame duplication in DNAJC5, encoding the cysteine-string protein alpha (CSPα). This variant leads to a duplication of the central core motif of the cysteine-string domain of CSPα and affects palmitoylation-dependent CSPα sorting in cultured neuronal cells similarly to two previously described CSPα variants, p.(Leu115Arg) and p.(Leu116del). Interestingly, the duplication was not detected initially by standard Sanger sequencing due to a preferential PCR amplification of the shorter wild-type allele and allelic dropout of the mutated DNAJC5 allele. It was also missed by subsequent whole-exome sequencing (WES). Its identification was facilitated by reanalysis of original WES data and modification of the PCR and Sanger sequencing protocols. Independently occurring variants in the genomic sequence of DNAJC5 encoding the cysteine-string domain of CSPα suggest that this region may be more prone to DNA replication errors and that insertions or duplications within this domain should be considered in unsolved ANCL cases.

γ-Aminobutyric acid receptor alpha 1 subunit loss of function causes genetic generalized epilepsy by impairing inhibitory network neurodevelopment.

OBJECTIVE: In humans, mutations of the γ-aminobutyric acid receptor subunit 1 (GABRA1) cause either mild or severe generalized epilepsy. Although these epilepsy-causing mutations have been shown to disrupt the receptor activity in vitro, their in vivo consequences on brain development and activity are not known. Here, we aim at unraveling the epileptogenesis mechanisms of GABRA1 loss of function. METHODS: We generated a gabra1-/- zebrafish mutant line displaying highly penetrant epileptic seizures. We sought to identify the underlying molecular mechanisms through unbiased whole transcriptomic assay of gabra1-/- larval brains. RESULTS: Interestingly, mutant fish show fully penetrant seizures at juvenile stages that accurately mimic tonic-clonic generalized seizures observed in patients. Moreover, highly penetrant seizures can be induced by light stimulation, thus providing us with the first zebrafish model in which evident epileptic seizures can be induced by nonchemical agents. Our transcriptomic assay identified misregulated genes in several pathways essential for correct brain development. More specifically, we show that the early development of the brain inhibitory network is specifically affected. Although the number of GABAergic neurons is not altered, we observed a drastic reduction in the number of inhibitory synapses and a decreased complexity of the GABAergic network. This is consistent with the disruption in expression of many genes involved in axon guidance and synapse formation. SIGNIFICANCE: Together with the role of GABA in neurodevelopment, our data identify a novel aspect of epileptogenesis, suggesting that the substratum of GABRA1-deficiency epilepsy is a consequence of early brain neurodevelopmental defects, in particular at the level of inhibitory network wiring.

Human copy number variants are enriched in regions of low mappability.

Copy number variants (CNVs) are known to affect a large portion of the human genome and have been implicated in many diseases. Although whole-genome sequencing (WGS) can help identify CNVs, most analytical methods suffer from limited sensitivity and specificity, especially in regions of low mappability. To address this, we use PopSV, a CNV caller that relies on multiple samples to control for technical variation. We demonstrate that our calls are stable across different types of repeat-rich regions and validate the accuracy of our predictions using orthogonal approaches. Applying PopSV to 640 human genomes, we find that low-mappability regions are approximately 5 times more likely to harbor germline CNVs, in stark contrast to the nearly uniform distribution observed for somatic CNVs in 95 cancer genomes. In addition to known enrichments in segmental duplication and near centromeres and telomeres, we also report that CNVs are enriched in specific types of satellite and in some of the most recent families of transposable elements. Finally, using this comprehensive approach, we identify 3455 regions with recurrent CNVs that were missing from existing catalogs. In particular, we identify 347 genes with a novel exonic CNV in low-mappability regions, including 29 genes previously associated with disease.

The clinical impact of integration of magnetoencephalography in the presurgical workup for refractory nonlesional epilepsy.

OBJECTIVE: For patients with nonlesional refractory focal epilepsy (NLRFE), localization of the epileptogenic zone is more arduous, and intracranial electroencephalography (EEG) (icEEG) is frequently required. Planning for icEEG is dependent on combined data from multiple noninvasive modalities. We report the negative impact of lack of integration of magnetoencephalography (MEG) in the presurgical workup in NLRFE. METHODS: Observational MEG case series involving 31 consecutive patients with NLRFE in an academic epilepsy center. For various reasons, MEG data were not analyzed in a timely manner to be included in the decision-making process. The presumed impact of MEG was assessed retrospectively. RESULTS: Magnetoencephalography would have changed the initial management in 21/31 (68%) had MEG results been available by reducing the number of intracranial electrodes, modifying their position, allowing for direct surgery, canceling the intracranial study, or providing enough evidence to justify one. Good surgical outcome was achieved in 11 out of 17 patients who proceeded to epilepsy surgery. Nine out of eleven had MEG clusters corresponding to the resection area, and MEG findings would have allowed for direct surgery (avoiding icEEG) in 2/11. Six patients had poor outcome including three patients where MEG would have significantly changed the outcome by modifying the resection margin. Magnetoencephalography provided superior information in 3 patients where inadequate coverage precluded accurate mapping of the epileptogenic zone. CONCLUSION: In this single center retrospective study, MEG would have changed patient management, icEEG planning, and surgical outcome in a significant percentage of patients with NLRFE and should be considered in the presurgical workup in those patients.

Familial focal epilepsy with focal cortical dysplasia due to DEPDC5 mutations.

OBJECTIVE: The DEPDC5 (DEP domain-containing protein 5) gene, encoding a repressor of the mTORC1 signaling pathway, has recently emerged as a major gene mutated in familial focal epilepsies. We aimed to further extend the role of DEPDC5 to focal cortical dysplasias (FCDs). METHODS: Seven patients from 4 families with DEPDC5 mutations and focal epilepsy associated with FCD were recruited and investigated at the clinical, neuroimaging, and histopathological levels. The DEPDC5 gene was sequenced from genomic blood and brain DNA. RESULTS: All patients had drug-resistant focal epilepsy, 5 of them underwent surgery, and 1 had a brain biopsy. Electroclinical phenotypes were compatible with FCD II, although magnetic resonance imaging (MRI) was typical in only 4 cases. Histopathology confirmed FCD IIa in 2 patients (including 1 MRI-negative case) and showed FCD I in 2 other patients, and remained inconclusive in the last 2 patients. Three patients were seizure-free postsurgically, and 1 had a worthwhile improvement. Sequencing of blood DNA revealed truncating DEPDC5 mutations in all 4 families; 1 mutation was found to be mosaic in an asymptomatic father. A brain somatic DEPDC5 mutation was identified in 1 patient in addition to the germline mutation. INTERPRETATION: Germline, germline mosaic, and brain somatic DEPDC5 mutations may cause epilepsy associated with FCD, reinforcing the link between mTORC1 pathway and FCDs. Similarly to other mTORopathies, a "2-hit" mutational model could be responsible for cortical lesions. Our study also indicates that epilepsy surgery is a valuable alternative in the treatment of drug-resistant DEPDC5-positive focal epilepsies, even if the MRI is unremarkable.

Two definite cases of sudden unexpected death in epilepsy in a family with a DEPDC5 mutation.

The DEPDC5 gene (OMIM #614191), mapped to 22q12.2-q12.3, encodes the DEP domain-containing protein 5. DEPDC5 has been associated with a variety of familial epilepsies, including familial focal epilepsy with variable foci, autosomal dominant nocturnal frontal lobe epilepsy, familial temporal lobe epilepsy, epileptic spasms, and cortical dysplasia.(1-4) Notably, DEPDC5 has never been linked to increased risk of sudden unexpected death in epilepsy (SUDEP). We report a family with epilepsy due to DEPDC5 mutation and 2 definite cases of SUDEP within this family.

Cathepsin F mutations cause Type B Kufs disease, an adult-onset neuronal ceroid lipofuscinosis.

Kufs disease, an adult-onset neuronal ceroid lipofuscinosis, is challenging to diagnose and genetically heterogeneous. Mutations in CLN6 were recently identified in recessive Kufs disease presenting as progressive myoclonus epilepsy (Type A), whereas the molecular basis of cases presenting with dementia and motor features (Type B) is unknown. We performed genome-wide linkage mapping of two families with recessive Type B Kufs disease and identified a single region on chromosome 11 to which both families showed linkage. Exome sequencing of five samples from the two families identified homozygous and compound heterozygous missense mutations in CTSF within this linkage region. We subsequently sequenced CTSF in 22 unrelated individuals with suspected recessive Kufs disease, and identified an additional patient with compound heterozygous mutations. CTSF encodes cathepsin F, a lysosomal cysteine protease, dysfunction of which is a highly plausible candidate mechanism for a storage disorder like ceroid lipofuscinosis. In silico modeling suggested the missense mutations would alter protein structure and function. Moreover, re-examination of a previously published mouse knockout of Ctsf shows that it recapitulates the light and electron-microscopic pathological features of Kufs disease. Although CTSF mutations account for a minority of cases of type B Kufs, CTSF screening should be considered in cases with early-onset dementia and may avoid the need for invasive biopsies.

Exome sequencing identifies FUS mutations as a cause of essential tremor.

Essential tremor (ET) is a common neurodegenerative disorder that is characterized by a postural or motion tremor. Despite a strong genetic basis, a gene with rare pathogenic mutations that cause ET has not yet been reported. We used exome sequencing to implement a simple approach to control for misdiagnosis of ET, as well as phenocopies involving sporadic and senile ET cases. We studied a large ET-affected family and identified a FUS p.Gln290(∗) mutation as the cause of ET in this family. Further screening of 270 ET cases identified two additional rare missense FUS variants. Functional considerations suggest that the pathogenic effects of ET-specific FUS mutations are different from the effects observed when FUS is mutated in amyotrophic lateral sclerosis cases; we have shown that the ET FUS nonsense mutation is degraded by the nonsense-mediated-decay pathway, whereas amyotrophic lateral sclerosis FUS mutant transcripts are not.

Kufs disease, the major adult form of neuronal ceroid lipofuscinosis, caused by mutations in CLN6.

The molecular basis of Kufs disease is unknown, whereas a series of genes accounting for most of the childhood-onset forms of neuronal ceroid lipofuscinosis (NCL) have been identified. Diagnosis of Kufs disease is difficult because the characteristic lipopigment is largely confined to neurons and can require a brain biopsy or autopsy for final diagnosis. We mapped four families with Kufs disease for whom there was good evidence of autosomal-recessive inheritance and found two peaks on chromosome 15. Three of the families were affected by Kufs type A disease and presented with progressive myoclonus epilepsy, and one was affected by type B (presenting with dementia and motor system dysfunction). Sequencing of a candidate gene in one peak shared by all four families identified no mutations, but sequencing of CLN6, found in the second peak and shared by only the three families affected by Kufs type A disease, revealed pathogenic mutations in all three families. We subsequently sequenced CLN6 in eight other families, three of which were affected by recessive Kufs type A disease. Mutations in both CLN6 alleles were found in the three type A cases and in one family affected by unclassified Kufs disease. Mutations in CLN6 are the major cause of recessive Kufs type A disease. The phenotypic differences between variant late-infantile NCL, previously found to be caused by CLN6, and Kufs type A disease are striking; there is a much later age at onset and lack of visual involvement in the latter. Sequencing of CLN6 will provide a simple diagnostic strategy in this disorder, in which definitive identification usually requires invasive biopsy.

Value of 3.0 T MR imaging in refractory partial epilepsy and negative 1.5 T MRI.

BACKGROUND: High-field 3.0 T MR scanners provide an improved signal-to-noise ratio which can be translated in higher image resolution, possibly allowing critical detection of subtle epileptogenic lesions missed on standard-field 1.0-1.5 T MRIs. In this study, the authors explore the potential value of re-imaging at 3.0 T patients with refractory partial epilepsy and negative 1.5 T MRI. METHODS: We retrospectively identified all patients with refractory partial epilepsy candidate for surgery who had undergone a 3.0 T MR study after a negative 1.5 T MR study. High-field 3.0 T MRIs were reviewed qualitatively by neuroradiologists experienced in interpreting epilepsy studies with access to clinical information. Relevance and impact on clinical management were assessed by an epileptologist. RESULTS: Between November 2006 and August 2009, 36 patients with refractory partial epilepsy candidate for surgery underwent 3.0 T MR study after a 1.5 T MR study failed to disclose a relevant epileptogenic lesion. A potential lesion was found only in two patients (5.6%, 95% CI: 1.5-18.1%). Both were found to have hippocampal atrophy congruent with other presurgical localization techniques which resulted in omission of an invasive EEG study and direct passage to surgery. CONCLUSIONS: The frequency of detection of a new lesion by re-imaging at 3.0 T patients with refractory partial epilepsy candidate for surgery was found to be low, but seems to offer the potential of a significant clinical impact for selected patients. This finding needs to be validated in a prospective controlled study.

Microsurgery of epileptic foci in the insular region.

OBJECT: The insular region has long been neglected in the investigation and treatment of refractory epilepsy. Surgery in the insular region is rarely performed because of the risk of injury to the opercula, the arteries transiting on the surface of the insula, and the deep structures such as the basal ganglia and the internal capsule. This study was undertaken to report the results of insular surgery using modern microsurgical techniques in patients with epilepsy. METHODS: The authors performed a retrospective study of cases involving patients who underwent surgery for insular lesions associated with epilepsy over the last 10 years. In the majority of patients, intracranial electrodes were implanted with neuronavigation guidance to confirm the localization of the epileptic foci. RESULTS: Nine patients underwent insular surgery: 7 for refractory epilepsy with no tumor and 2 for tumors associated with seizures. Four of the resections were performed in the left hemisphere. After an average follow-up of 54 months (range 14-122 months), Engel Class IA outcome had been achieved in 6 of 7 cases in the Epilepsy Surgery Group. The remaining patient had an Engel Class III outcome after partial insular resection but later became seizurefree (Engel Class IA) following insular Gamma Knife surgery. Postoperatively, the majority of patients suffered from minor reversible hemipareses that disappeared completely within a few months. There was no surgical mortality. CONCLUSIONS: Insular surgery is both safe and beneficial when it is well planned and performed with modern microsurgical techniques and good anatomical knowledge. Insulectomy is associated with little permanent morbidity and a high rate of seizure control. To the authors' knowledge, this is the first series of insulectomies predominantly performed for refractory epilepsy since those performed by Penfield.

Revisiting the role of the insula in refractory partial epilepsy.

PURPOSE: Recent evidence suggesting that some epilepsy surgery failures could be related to unrecognized insular epilepsy have led us to lower our threshold to sample the insula with intracerebral electrodes. In this study, we report our experience resulting from this change in strategy. METHODS: During the period extending from October 2004 to June 2007, 18 patients had an intracranial study including 10 with insular coverage. The decision to sample the insula with intracerebral electrodes was made in the context of (1) nonlesional parietal lobe-like epilepsy; (2) nonlesional frontal lobe-like epilepsy; (3) nonlesional temporal lobe-like epilepsy; and (4) atypical temporal lobe-like epilepsy. RESULTS: Intracerebral recordings confirmed the presence of insular lobe seizures in four patients. Cortical stimulation performed in 9 of 10 patients with insular electrodes elicited, in decreasing order of frequency, somatosensory, viscerosensory, motor, auditory, vestibular, and speech symptoms. DISCUSSION: Our results suggest that insular cortex epilepsy may mimic temporal, frontal, and parietal lobe epilepsies and that a nonnegligeable proportion of surgical candidates with drug-resistant epilepsy have an epileptogenic zone that involves the insula.

Disruption of AP1S1, causing a novel neurocutaneous syndrome, perturbs development of the skin and spinal cord.

Adaptor protein (AP) complexes regulate clathrin-coated vesicle assembly, protein cargo sorting, and vesicular trafficking between organelles in eukaryotic cells. Because disruption of the various subunits of the AP complexes is embryonic lethal in the majority of cases, characterization of their function in vivo is still lacking. Here, we describe the first mutation in the human AP1S1 gene, encoding the small subunit sigma1A of the AP-1 complex. This founder splice mutation, which leads to a premature stop codon, was found in four families with a unique syndrome characterized by mental retardation, enteropathy, deafness, peripheral neuropathy, ichthyosis, and keratodermia (MEDNIK). To validate the pathogenic effect of the mutation, we knocked down Ap1s1 expression in zebrafish using selective antisens morpholino oligonucleotides (AMO). The knockdown phenotype consisted of perturbation in skin formation, reduced pigmentation, and severe motility deficits due to impaired neural network development. Both neural and skin defects were rescued by co-injection of AMO with wild-type (WT) human AP1S1 mRNA, but not by co-injecting the truncated form of AP1S1, consistent with a loss-of-function effect of this mutation. Together, these results confirm AP1S1 as the gene responsible for MEDNIK syndrome and demonstrate a critical role of AP1S1 in development of the skin and spinal cord.

SPG4 founder effect in French Canadians with hereditary spastic paraplegia.

BACKGROUND: The most common cause of autosomal dominant Hereditary Spastic Paraplegia (HSP) is mutations in the SPG4 gene. We have previously identified novel SPG4 mutations in a collection of North American families including the c.G1801A mutation present in two families from Quebec. The aim of this study is to estimate the frequency of the c.G1801A mutation in the French Canadian (FC) population and to determine whether this mutation originates from a common ancestor. METHODS: We collected and sequenced exon 15 in probands of 37 families. Genotypes of markers flanking the SPG4 gene were used to construct haplotypes in five families. Clinical information was reviewed by a neurologist with expertise in HSP. RESULTS: We have identified three additional unrelated families with the c.G1801A mutation and haplotype analysis revealed that all five families share a common ancestor. The mutation is present in 7% of all our FC families and explains half of our spastin linked FC families. The phenotype associated with the c.G1801A genotype is pure HSP with bladder involvement. CONCLUSION: In this study we have determined that the relative frequency of the c.G1801A mutation in our FC collection is 7%, and approximately 50% in the spastin positive FC group. This mutation is the most common HSP mutation identified in this population to date and is suggestive of a founder effect in Quebec.