A missense mutation in the neuronal nicotinic acetylcholine receptor alpha 4 subunit is associated with autosomal dominant nocturnal frontal lobe epilepsy.

Epilepsy affects at least 2% of the population at some time in their lives. The epilepsies are a heterogeneous group of disorders, many with an inherited component. Although specific genes have been identified in a few rare diseases causing seizures as part of a more diffuse brain disorder, the molecular pathology of the common idiopathic epilepsies is still unknown. Linkage has been reported for some generalised epilepsy syndromes, but only very recently for familial partial epilepsy syndromes. Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a partial epilepsy causing frequent, violent, brief seizures at night, usually beginning in childhood. The gene for ADNFLE maps to chromosome 20q13.2-q13.3 in one large Australian kindred. The neuronal nicotinic acetylcholine receptor alpha 4 subunit (CHRNA4) maps to the same region of 20q (ref. 12) and the gene is expressed in all layers of the frontal cortex. We screened affected family members for mutations within CHRNA4 and found a missense mutation that replaces serine with phenylalanine at codon 248, a strongly conserved amino acid residue in the second transmembrane domain. The mutation is present in all 21 available affected family members and in four obligate carriers, but not in 333 healthy control subjects.

Localization of a gene for autosomal dominant nocturnal frontal lobe epilepsy to chromosome 20q 13.2.

The epilepsies comprise a group of syndromes that are divided into generalized and partial (focal) types. Familial occurrence has long been recognized but progress in mapping epilepsy genes has been slow except for rare cases where the inheritance is easily determined from classical genetic studies. Linkage is established for three generalized syndromes: the EBN1 and EBN2 genes for benign familial neonatal convulsions (BFNC) map to chromosomes 20q and 8q (refs 2-5), the EPM1 gene for Unverricht-Lundborg disease maps to 21q (ref. 6) and the gene for the northern epilepsy syndrome maps to 8p (ref. 7). A claim for linkage of the EJM1 gene for the common generalized syndrome of juvenile myoclonic epilepsy to 6p is currently in dispute. Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) was recently described in five families. We now report the chromosomal assignment, to 20q13.2, for the gene for ADNFLE in one large Australian kindred with 27 affected individuals spanning six generations.

Febrile seizures and generalized epilepsy associated with a mutation in the Na+-channel beta1 subunit gene SCN1B.

Febrile seizures affect approximately 3% of all children under six years of age and are by far the most common seizure disorder. A small proportion of children with febrile seizures later develop ongoing epilepsy with afebrile seizures. Segregation analysis suggests the majority of cases have complex inheritance but rare families show apparent autosomal dominant inheritance. Two putative loci have been mapped (FEB1 and FEB2), but specific genes have not yet been identified. We recently described a clinical subset, termed generalized epilepsy with febrile seizures plus (GEFS+), in which many family members have seizures with fever that may persist beyond six years of age or be associated with afebrile generalized seizures. We now report linkage, in another large GEFS+ family, to chromosome region 19q13.1 and identification of a mutation in the voltage-gated sodium (Na+)-channel beta1 subunit gene (SCN1B). The mutation changes a conserved cysteine residue disrupting a putative disulfide bridge which normally maintains an extracellular immunoglobulin-like fold. Co-expression of the mutant beta1 subunit with a brain Na+-channel alpha subunit in Xenopus laevis oocytes demonstrates that the mutation interferes with the ability of the subunit to modulate channel-gating kinetics consistent with a loss-of-function allele. This observation develops the theme that idiopathic epilepsies are a family of channelopathies and raises the possibility of involvement of other Na+-channel subunit genes in febrile seizures and generalized epilepsies with complex inheritance patterns.

Mutant GABA(A) receptor gamma2-subunit in childhood absence epilepsy and febrile seizures.

Epilepsies affect at least 2% of the population at some time in life, and many forms have genetic determinants. We have found a mutation in a gene encoding a GABA(A) receptor subunit in a large family with epilepsy. The two main phenotypes were childhood absence epilepsy (CAE) and febrile seizures (FS). There is a recognized genetic relationship between FS and CAE, yet the two syndromes have different ages of onset, and the physiology of absences and convulsions is distinct. This suggests the mutation has age-dependent effects on different neuronal networks that influence the expression of these clinically distinct, but genetically related, epilepsy phenotypes. We found that the mutation in GABRG2 (encoding the gamma2-subunit) abolished in vitro sensitivity to diazepam, raising the possibility that endozepines do in fact exist and have a physiological role in preventing seizures.

Generation of an iPSC line (FINi001-A) from a girl with developmental and epileptic encephalopathy due to a heterozygous gain-of-function p.R1882Q variant in the voltage-gated sodium channel Nav1.2 protein encoded by the SCN2A gene.

A range of epilepsies, including the most severe group of developmental and epileptic encephalopathies (DEEs), are caused by gain-of-function variants in voltage-gated channels. Here we report the generation and characterisation of an iPSC cell line from the fibroblasts of a girl with early infantile DEE carrying heterozygous missense gain-of-function mutation (R1882Q) in Nav1.2(SCN2A) protein, using transient transfection with a single mRNA molecule. The established iPSC line displays typical human primed pluripotent stem cell characteristics: typical colony morphology and robust expression of pluripotency-associated marker genes, ability to give rise to derivatives of all three embryonic germ layers, and normal karyotype without any SNP array-detectable copy number variations. We anticipate that this iPSC line will be useful for the development of neuronal hyperactivity-caused human stem cell-based DEE models, advancing both understanding and potential therapy development for this debilitating condition.

Distinctive Brain Malformations in Zhu-Tokita-Takenouchi-Kim Syndrome.

BACKGROUND AND PURPOSE: Zhu-Tokita-Takenouchi-Kim syndrome is a severe multisystem malformation disorder characterized by developmental delay and a diverse array of congenital abnormalities. However, these currently identified phenotypic components provide limited guidance in diagnostic situations, due to both the nonspecificity and variability of these features. Here we report a case series of 7 individuals with a molecular diagnosis of Zhu-Tokita-Takenouchi-Kim syndrome, 5 ascertained by their presentation with the neuronal migration disorder, periventricular nodular heterotopia. MATERIALS AND METHODS: Individuals with a molecular diagnosis of Zhu-Tokita-Takenouchi-Kim syndrome were recruited from 2 sources, a high-throughput sequencing study of individuals with periventricular nodular heterotopia or from clinical diagnostic sequencing studies. We analyzed available brain MR images of recruited individuals to characterize periventricular nodular heterotopia distribution and to identify the presence of any additional brain abnormalities. RESULTS: Pathogenic variants in SON, causative of Zhu-Tokita-Takenouchi-Kim syndrome, were identified in 7 individuals. Brain MR images from these individuals were re-analyzed. A characteristic set of imaging anomalies in addition to periventricular nodular heterotopia was identified, including the elongation of the pituitary stalk, cerebellar enlargement with an abnormally shaped posterior fossa, rounding of the caudate nuclei, hippocampal malformations, and cortical anomalies including polymicrogyria or dysgyria. CONCLUSIONS: The recurrent neuroradiologic changes identified here represent an opportunity to guide diagnostic formulation of Zhu-Tokita-Takenouchi-Kim syndrome on the basis of brain MR imaging evaluation.

Unclassified white matter disorders: A diagnostic journey requiring close collaboration between clinical and laboratory services.

BACKGROUND: Next generation sequencing studies have revealed an ever-increasing number of causes for genetic disorders of central nervous system white matter. A substantial number of disorders are identifiable from their specific pattern of biochemical and/or imaging findings for which single gene testing may be indicated. Beyond this group, the causes of genetic white matter disorders are unclear and a broader approach to genomic testing is recommended. AIM: This study aimed to identify the genetic causes for a group of individuals with unclassified white matter disorders with suspected genetic aetiology and highlight the investigations required when the initial testing is non-diagnostic. METHODS: Twenty-six individuals from 22 families with unclassified white matter disorders underwent deep phenotyping and genome sequencing performed on trio, or larger, family groups. Functional studies and transcriptomics were used to resolve variants of uncertain significance with potential clinical relevance. RESULTS: Causative or candidate variants were identified in 15/22 (68.2%) families. Six of the 15 implicated genes had been previously associated with white matter disease (COL4A1, NDUFV1, SLC17A5, TUBB4A, BOLA3, DARS2). Patients with variants in the latter two presented with an atypical phenotype. The other nine genes had not been specifically associated with white matter disease at the time of diagnosis and included genes associated with monogenic syndromes, developmental disorders, and developmental and epileptic encephalopathies (STAG2, LSS, FIG4, GLS, PMPCA, SPTBN1, AGO2, SCN2A, SCN8A). Consequently, only 46% of the diagnoses would have been made via a current leukodystrophy gene panel test. DISCUSSION: These results confirm the importance of broad genomic testing for patients with white matter disorders. The high diagnostic yield reflects the integration of deep phenotyping, whole genome sequencing, trio analysis, functional studies, and transcriptomic analyses. CONCLUSIONS: Genetic white matter disorders are genetically and phenotypically heterogeneous. Deep phenotyping together with a range of genomic technologies underpin the identification of causes of unclassified white matter disease. A molecular diagnosis is essential for prognostication, appropriate management, and accurate reproductive counseling.

Detection of cryptic pathogenic copy number variations and constitutional loss of heterozygosity using high resolution SNP microarray analysis in 117 patients referred for cytogenetic analysis and impact on clinical practice.

BACKGROUND: Microarray genome analysis is realising its promise for improving detection of genetic abnormalities in individuals with mental retardation and congenital abnormality. Copy number variations (CNVs) are now readily detectable using a variety of platforms and a major challenge is the distinction of pathogenic from ubiquitous, benign polymorphic CNVs. The aim of this study was to investigate replacement of time consuming, locus specific testing for specific microdeletion and microduplication syndromes with microarray analysis, which theoretically should detect all known syndromes with CNV aetiologies as well as new ones. METHODS: Genome wide copy number analysis was performed on 117 patients using Affymetrix 250K microarrays. RESULTS: 434 CNVs (195 losses and 239 gains) were found, including 18 pathogenic CNVs and 9 identified as "potentially pathogenic". Almost all pathogenic CNVs were larger than 500 kb, significantly larger than the median size of all CNVs detected. Segmental regions of loss of heterozygosity larger than 5 Mb were found in 5 patients. CONCLUSIONS: Genome microarray analysis has improved diagnostic success in this group of patients. Several examples of recently discovered "new syndromes" were found suggesting they are more common than previously suspected and collectively are likely to be a major cause of mental retardation. The findings have several implications for clinical practice. The study revealed the potential to make genetic diagnoses that were not evident in the clinical presentation, with implications for pretest counselling and the consent process. The importance of contributing novel CNVs to high quality databases for genotype-phenotype analysis and review of guidelines for selection of individuals for microarray analysis is emphasised.

Periventricular heterotopia: an X-linked dominant epilepsy locus causing aberrant cerebral cortical development.

Periventricular heterotopia (PH) involves dramatic malformations of the human cerebral cortex. Here we show that PH is closely linked to markers in distal Xq28 (maximal two-point lod score = 4.77 for F8C at theta = 0; maximal multipoint lod score = 5.37), so that affected females are obligatory mosaics for the mutation; that PH is lethal to at least some affected males; that PH malformations consist of well-differentiated cortical neurons filling the adult subependymal zone; and that individuals with PH are at high risk for epilepsy, though they have no other neurological or external stigmata. The PH gene may represent an important epilepsy susceptibility locus in addition to playing a key role in normal cortical development.

Mutations in filamin 1 prevent migration of cerebral cortical neurons in human periventricular heterotopia.

Long-range, directed migration is particularly dramatic in the cerebral cortex, where postmitotic neurons generated deep in the brain migrate to form layers with distinct form and function. In the X-linked dominant human disorder periventricular heterotopia (PH), many neurons fail to migrate and persist as nodules lining the ventricular surface. Females with PH present with epilepsy and other signs, including patent ductus arteriosus and coagulopathy, while hemizygous males die embryonically. We have identified the PH gene as filamin 1 (FLN1), which encodes an actin-cross-linking phosphoprotein that transduces ligand-receptor binding into actin reorganization, and which is required for locomotion of many cell types. FLN1 shows previously unrecognized, high-level expression in the developing cortex, is required for neuronal migration to the cortex, and is essential for embryogenesis.

Deletions or duplications in KCNQ2 can cause benign familial neonatal seizures.

BACKGROUND: Benign familial neonatal seizures are most often caused by mutations in the voltage-gated potassium channel subunit gene KCNQ2. More than 60 mutations have been described in BFNS families, approximately half of which lead to protein truncation. The hypothesis of this study was that deletion or duplication of >or=1 exons of KCNQ2 could cause BFNS in cases without coding or splicing mutations. METHODS: Multiplex ligation-dependent probe amplification (MLPA) was used to test a group of 21 unrelated patients with clinical features consistent with either BFNS, benign familial neonatal-infantile seizures or sporadic neonatal seizures, for exonic deletions and duplications. RESULTS: Three deletions and one duplication mutation were identified in four familial cases and cascade testing of their available family members showed that the mutations segregated with the phenotype in each family. The junction fragment for one of the deletions was amplified by PCR and sequenced to characterise the breakpoint and verify that a deletion had occurred. CONCLUSIONS: Submicroscopic deletions or duplications of KCNQ2 are seen in a significant proportion of BFNS families: four of nine (44%) cases previously testing negative for coding or splice site mutation by sequencing KCNQ2 and KCNQ3. MLPA is an efficient second-tier testing strategy for KCNQ2 to identify pathogenic intragenic mutations not detectable by conventional DNA sequencing methods.

NEDD4-2 as a potential candidate susceptibility gene for epileptic photosensitivity.

Photosensitive seizures occur most commonly in childhood and adolescence, usually as a manifestation of complex idiopathic generalized epilepsies (IGEs). Molecular mechanisms underlying this condition are yet to be determined because no susceptibility genes have been identified. The NEDD4-2 (Neuronally Expressed Developmentally Downregulated 4) gene encodes a ubiquitin protein ligase proposed to regulate cell surface levels of several ion channels, receptors and transporters involved in regulating neuronal excitability, including voltage-gated sodium channels (VGSCs), the most clinically relevant of the epilepsy genes. The regulation of NEDD4-2 in vivo involves complex interactions with accessory proteins in a cell type specific manner. We screened NEDD4-2 for mutations in a cohort of 253 families with IGEs. We identified three NEDD4-2 missense changes in highly conserved residues; S233L, E271A and H515P in families with photosensitive generalized epilepsy. The NEDD4-2 variants were as effective as wild-type NEDD4-2 in downregulating the VGSC subtype Na(v)1.2 when assessed in the Xenopus oocyte heterologous expression system showing that the direct interaction with the ion channel was not altered by these variants. These data raise the possibility that photosensitive epilepsy may arise from defective interaction of NEDD4-2 with as yet unidentified accessory or target proteins.

Generalized epilepsy with febrile seizures plus-associated sodium channel beta1 subunit mutations severely reduce beta subunit-mediated modulation of sodium channel function.

Two novel mutations (R85C and R85H) on the extracellular immunoglobulin-like domain of the sodium channel beta1 subunit have been identified in individuals from two families with generalized epilepsy with febrile seizures plus (GEFS+). The functional consequences of these two mutations were determined by co-expression of the human brain NaV1.2 alpha subunit with wild type or mutant beta1 subunits in human embryonic kidney (HEK)-293T cells. Patch clamp studies confirmed the regulatory role of beta1 in that relative to NaV1.2 alone the NaV1.2+beta1 currents had right-shifted voltage dependence of activation, fast and slow inactivation and reduced use dependence. In addition, the NaV1.2+beta1 current entered fast inactivation slightly faster than NaV1.2 channels alone. The beta1(R85C) subunit appears to be a complete loss of function in that none of the modulating effects of the wild type beta1 were observed when it was co-expressed with NaV1.2. Interestingly, the beta1(R85H) subunit also failed to modulate fast kinetics, however, it shifted the voltage dependence of steady state slow inactivation in the same way as the wild type beta1 subunit. Immunohistochemical studies revealed cell surface expression of the wild type beta1 subunit and undetectable levels of cell surface expression for both mutants. The functional studies suggest association of the beta1(R85H) subunit with the alpha subunit where its influence is limited to modulating steady state slow inactivation. In summary, the mutant beta1 subunits essentially fail to modulate alpha subunits which could increase neuronal excitability and underlie GEFS+ pathogenesis.

The role of genetics and ethnicity in epilepsy management.

Recent exciting developments in epilepsy genetics have led to significant insights into the mechanisms underlying seizure disorders. Success in epilepsy genetics research to date has resulted from identification of genes responsible for rare monogenic disorders, the majority encoding either voltage- or ligand-gated ion channels. For some conditions, such as benign familial neonatal seizures, an understanding of the underlying genetics is helpful in predicting prognosis. However, for other disorders, such as autosomal dominant nocturnal frontal lobe epilepsy, phenotypic severity is determined by factors other than the major dominant nicotinic subunit mutation found in some families. Further complexity arises when single-gene mutations give rise to heterogeneous phenotypes, as typically occur with generalized epilepsy with febrile seizures plus. Another area of increasing genetic endeavour, pharmacogenetics will allow tailoring of antiepileptic medication for each patient. Pharmacogenetics explores genetic polymorphisms in genes coding for drug-metabolizing enzymes, receptors and transporters. Polymorphisms have been identified that result in marked ethnic and interindividual differences in response to treatment. With further understanding of the impact of these differences, pharmacogenetic screening is likely to guide the management of epilepsy in the future.

Phenotypic expression of benign familial neonatal convulsions linked to chromosome 20.

OBJECTIVES: To determine whether the syndrome of benign familial neonatal convulsions in a large family was linked to markers on chromosome 20q and to study the seizure patterns in affected individuals. DESIGN: A clinical and molecular biologic study of a single large family in which the probands were identical twins with benign familial neonatal convulsions. PATIENTS: Thirteen living affected family members and 27 living unaffected family members were evaluated. RESULTS: Multipoint linkage analysis with use of the chromosome 20q markers CMM6 and RMR6 gave a maximum lod score of 3.13 at theta = 0.063, indicating linkage in this family. Of the 13 affected members, 10 had known neonatal seizures. Four subjects had febrile seizures, of whom only two had known neonatal seizures. Two members had afebrile seizures later, one of whom had not previously suffered neonatal or febrile seizures. CONCLUSION: The phenotypic heterogeneity in this family, with an epilepsy syndrome determined by a single gene, was striking. This suggests that molecular genetic approaches to the common forms of idiopathic epilepsy, involving patients with clinically similar phenotypes from unrelated families, may be inappropriate.

Autosomal dominant nocturnal frontal lobe epilepsy: demonstration of focal frontal onset and intrafamilial variation.

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a newly recognized autosomal dominant partial epilepsy. We studied seizure localization and intrafamilial variation using video-EEG monitoring (VEM) and functional neuroimaging in two pairs of subjects from unrelated families. The clinical features of seizures were similar from seizure to seizure in each individual, but varied between individuals. As is often found in frontal lobe epilepsies, ictal EEG localization was imprecise in three of four cases. One patient showed a consistent left fronto-polar onset that was corroborated by congruent focal hypometabolism on interictal PET and focal hyperperfusion on ictal single photon emission computed tomography (SPECT). A second case studied with ictal SPECT showed a right parasagittal, midfrontal focus. We conclude that this autosomal dominant epilepsy syndrome, which in one of the two families was due to a known neuronal nicotinic acetylcholine receptor mutation, causes frontal lobe foci that are unilateral and in variable locations in different individuals.

Generalized epilepsy with febrile seizures plus: mutation of the sodium channel subunit SCN1B.

Generalized epilepsy with febrile seizures plus (GEFS(+)) is an important childhood genetic epilepsy syndrome with heterogeneous phenotypes, including febrile seizures (FS) and generalized epilepsies of variable severity. Forty unrelated GEFS(+) and FS patients were screened for mutations in the sodium channel beta-subunits SCN1B and SCN2B, and the second GEFS(+) family with an SCN1B mutation is described here. The family had 19 affected individuals: 16 with typical GEFS(+) phenotypes and three with other epilepsy phenotypes. Site-specific mutation within SCN1B remains a rare cause of GEFS(+), and the authors found no evidence to implicate SCN2B in this syndrome.

Motor cortex localization using functional MRI and transcranial magnetic stimulation.

OBJECTIVE: Congenital brain lesions producing focal seizures may be accompanied by reorganization of the areas responsible for motor and sensory functions within the brain due to a phenomenon that has been termed "neuronal plasticity." This can be studied using functional MRI (fMRI) and transcranial magnetic stimulation (TMS). Using either method, the motor cortex can be localized noninvasively, but to date there have been few studies correlating the level of agreement between the two techniques. METHODS: We used fMRI and TMS to localize the motor cortex in a young woman with intractable focal seizures, congenital left arm weakness, and a dysplastic right hemisphere on MRI. RESULTS: There was excellent agreement in the localization of motor representation for each hand. Both were predominantly located in the left hemisphere. fMRI also showed an area of posterior activation in the right hemisphere, but there was no evidence of descending corticospinal projections from this site using TMS, direct cortical stimulation, and Wada testing. CONCLUSIONS: Functional MRI (fMRI) and transcranial magnetic stimulation (TMS) were successfully used to localize cortical motor function before epilepsy surgery. Each technique demonstrated migration of motor function for the left hand to the left motor cortex. After resection of the dysplastic right precentral gyrus there was no permanent increase in weakness or disability. The two techniques are complementary; fMRI indicates all cortical areas activated by the motor task, whereas TMS identifies only those areas giving rise to corticospinal projections.

Genetic linkage studies in familial frontal epilepsy: exclusion of the human chromosome regions homologous to the El-1 mouse locus.

Familial frontal epilepsy has been recently described in six pedigrees. All families reported show autosomal dominant inheritance with incomplete penetrance. Affected individuals develop predominantly nocturnal seizures with frontal lobe semiology. In 1959, a genetic mouse model for partial epilepsy, the El mouse, was reported. In the El mouse, a major seizure susceptibility gene, El-1, segregates in an autosomal dominant fashion and has been localized to a region distal to the centromere of mouse ch 9. Comparative genetic maps between man and mouse have been used to predict the location of several human disease genes. The El-1 locus in the mouse is homologous to human chromosomes 3p23-p21.2, 3p11.2-q11.2, 3q21-q25.3, 6p12-q12 and 15q24. Polymorphic microsatellite markers covering these candidate regions were used for genotyping individuals in the three larger families ascertained, one of which is French-Canadian and two are Australian. Significant negative two-point and multipoint lod scores were obtained separately for each family, thus excluding linkage with the candidate regions on chromosomes 3, 6 and 15.

Epilepsies with single gene inheritance.

Single gene disorders offer the best opportunity for identification of genetic linkage and of abnormal genes. Epilepsies with single gene inheritance include symptomatic epilepsies where there is associated diffuse brain dysfunction, and idiopathic epilepsies where seizures are the major neurological abnormality. There are over 200 single gene symptomatic epilepsies; most are rare. Gene identification has been achieved in a number of these conditions but these important advances have not yet led to a better understanding of epileptogenesis, because of the associated brain disease. Idiopathic single gene epilepsies include benign familial neonatal convulsions, where genetic linkage to chromosomes 20q and 8q has been found in different families, and benign familial infantile convulsions where linkage is presently unknown. Recently, four autosomal dominant partial epilepsies have been described. In autosomal dominant nocturnal frontal lobe epilepsy a genetic defect in the alpha 4 subunit of the nicotinic acetylcholine receptor was found in one family. This is the first genetic defect described in an idiopathic epilepsy. The other three syndromes are autosomal dominant partial epilepsy with variable foci, autosomal dominant rolandic epilepsy with speech dyspraxia, and familial temporal lobe epilepsy. In the latter condition, linkage to chromosome 10q has been reported in one family, but the genetic defect is unknown. It is likely that other idiopathic single gene epilepsies will be identified. Molecular genetic study of these disorders is likely to lead to discovery of other epilepsy genes. This will lead to an improved understanding of human epileptogenesis with implications for clinical diagnosis, genetic counselling, pharmacological therapy and possibly prevention of epilepsy.