Evaluation of multiple putative risk alleles within the 15q13.3 region for genetic generalized epilepsy.

The chromosome 15q13.3 region has been implicated in epilepsy, intellectual disability and neuropsychiatric disorders, especially schizophrenia. Deficiency of the acetylcholine receptor gene CHRNA7 and the partial duplication, CHRFAM7A, may contribute to these phenotypes and we sought to comprehensively analyze these genes in genetic generalized epilepsy. We analyzed using DHPLC, Sanger sequencing and long range PCR, 174 probands with genetic generalized epilepsy with or without intellectual disability or psychosis, including 8 with the recurrent 15q13.3 microdeletion. We searched CHRNA7 and CHRFAM7A for single sequence variants, small copy number variants, and the common 2-bp deletion in CHRFAM7A. We identified two novel and one reported missense variants. The common 2-bp deletion was not enriched in patients compared to controls. Our data suggest that missense mutations in CHRNA7 contribute to complex inheritance in genetic generalized epilepsy in a similar fashion to the 15q13.3 microdeletion. They do not support a pathogenic role for the common 2-bp CHRFAM7A deletion.

PRIMA1 mutation: a new cause of nocturnal frontal lobe epilepsy.

OBJECTIVE: Nocturnal frontal lobe epilepsy (NFLE) can be sporadic or autosomal dominant; some families have nicotinic acetylcholine receptor subunit mutations. We report a novel autosomal recessive phenotype in a single family and identify the causative gene. METHODS: Whole exome sequencing data was used to map the family, thereby narrowing exome search space, and then to identify the mutation. RESULTS: Linkage analysis using exome sequence data from two affected and two unaffected subjects showed homozygous linkage peaks on chromosomes 7, 8, 13, and 14 with maximum LOD scores between 1.5 and 1.93. Exome variant filtering under these peaks revealed that the affected siblings were homozygous for a novel splice site mutation (c.93+2T>C) in the PRIMA1 gene on chromosome 14. No additional PRIMA1 mutations were found in 300 other NFLE cases. The c.93+2T>C mutation was shown to lead to skipping of the first coding exon of the PRIMA1 mRNA using a minigene system. INTERPRETATION: PRIMA1 is a transmembrane protein that anchors acetylcholinesterase (AChE), an enzyme hydrolyzing acetycholine, to membrane rafts of neurons. PRiMA knockout mice have reduction of AChE and accumulation of acetylcholine at the synapse; our minigene analysis suggests that the c.93+2T>C mutation leads to knockout of PRIMA1. Mutations with gain of function effects in acetylcholine receptor subunits cause autosomal dominant NFLE. Thus, enhanced cholinergic responses are the likely cause of the severe NFLE and intellectual disability segregating in this family, representing the first recessive case to be reported and the first PRIMA1 mutation implicated in disease.

Weight and fat distribution in patients taking valproate: a valproate-discordant gender-matched twin and sibling pair study.

OBJECTIVES: Chronic treatment with valproate (VPA) is commonly associated with weight gain, which potentially has important health implications, in particular increased central fat distribution. We utilized a VPA-discordant same-sex, twin and matched sibling pair study design to primarily examine for differences in fat distribution between patients with epilepsy treated with VPA compared to their matched twin or sibling control. Weight, blood pressure, and leptin levels were assessed. METHODS: Height, weight, waist and hip measurements, exercise, blood pressure (BP), and serum leptin levels were measured. Body composition was measured using dual-energy x-ray absorptiometry (DXA). Abdominal fat was expressed as a percentage of the abdominal region (AFat%); and of whole body fat (WBF); (AFat%WBF). Mean within-pair differences were assessed (VPA-user and nonuser). Restricted maximum likelihood (REML) linear mixed model analysis was fitted to examine associations of anthropometrics, zygosity, gender, menopausal status, VPA dose and duration, with weight and AFat%. RESULTS: We studied 19 pairs of VPA-discordant, gender-matched (five male, 14 female) twins and siblings. Mean (standard deviation, SD) duration of therapy for VPA users was 11.0 (7.4) years. There were no statistically significant within-pair differences in age, height, weight, body mass index (BMI), BP, leptin level, WBF, AFat%, or AFat%WBF. For pairs in which VPA-user was treated for >11 years there were statistically significant mean within-pair differences in AFat%, (+7.1%, p = 0.03, n = 10 pairs), mean BP (+11.0 mm Hg, p = 0.006, n = 8 pairs); but not in AFat%WBF. VPA duration was positively associated with weight (estimate +0.98 kg/per year of VPA, p = 0.03); VPA treatment duration and dose were not significantly associated with AFat%. SIGNIFICANCE: This study demonstrated a relationship between long-term VPA use and abdominal adiposity (AFat%), which could have significant health implications. We recommend ongoing monitoring of weight, BMI, and blood pressure for patients taking VPA.

Autosomal dominant vasovagal syncope: clinical features and linkage to chromosome 15q26.

OBJECTIVE: To establish the occurrence of an autosomal dominant form of vasovagal syncope (VVS) by detailed phenotyping of multiplex families and identification of the causative locus. METHODS: Patients with VVS and a family history of syncope were recruited. A standardized questionnaire was administered to all available family members and medical records were reviewed. Of 44 families recruited, 6 were suggestive of autosomal dominant inheritance. Genome-wide linkage was performed in family A using single nucleotide polymorphism genotyping microarrays. Targeted analysis of chromosome 15q26 with microsatellite markers was implemented in 4 families; 1 family was too small for analysis. RESULTS: Family A contained 30 affected individuals over 3 generations with a median onset of 8 to 9 years. The other families comprised 4 to 14 affected individuals. Affected individuals reported typical triggers of VVS (sight of blood, injury, medical procedures, prolonged standing, pain, frightening thoughts). The triggers varied considerably within the families. Significant linkage to chromosome 15q26 (logarithm of odds score 3.28) was found in family A. Linkage to this region was excluded in 2 medium-sized families but not in 2 smaller families. Sequence analysis of the candidate genes SLCO3A1, ST8SIA2, and NR2F2 within the linkage interval did not reveal any mutations. CONCLUSIONS: Familial VVS, inherited in an autosomal dominant manner, may not be rare and has similar features to sporadic VVS. The chromosome 15q26 locus in family A increases the susceptibility to VVS but does not predispose to a particular vasovagal trigger. Linkage analysis in the remaining families established likely genetic heterogeneity.

Genetics of febrile seizure subtypes and syndromes: a twin study.

PURPOSE: Febrile seizures (FS) are the most common seizure syndrome. A strong genetic component has been well established through family and twin studies; however, such studies have not examined the genetics of different FS types (simple, complex, febrile status epilepticus) and sub-syndromes (true FS, febrile seizures plus (FS+), 'FS with later epilepsy'). Here we used a community-based twin sample to analyze genetic factors within different FS subtypes and FS syndromes. METHODS: Twin pairs were ascertained from the twin database of the Epilepsy Research Centre. A retrospective chart review was conducted and follow-up attempted for all subjects. Casewise concordance values were calculated for the different subgroups and intra-pair variation was analyzed. KEY FINDINGS: One hundred and seventy-nine twin pairs with FS were identified. Overall casewise concordance for FS in monozygotic (MZ) twins (0.62) was greater than in dizygotic (DZ) twins (0.16, p<0.0001). A greater concordance amongst MZ pairs than DZ twin pairs was also observed for all FS subtypes and FS sub-syndromes, particularly in twins with FS+. Within concordant MZ pairs, we did not observe the co-occurrence of FS and FS+. SIGNIFICANCE: These results suggest a strong genetic contribution to different FS subtypes and sub-syndromes. They also support the existence of distinct genetic factors for different FS subtypes and sub-syndromes, especially FS+. This information is important for the strategic planning of next generation sequencing studies of febrile seizures.

Early onset absence epilepsy: 1 in 10 cases is caused by GLUT1 deficiency.

Glucose transporter 1 (GLUT1) deficiency caused by mutations of SLC2A1 is an increasingly recognized cause of genetic generalized epilepsy. We previously reported that >10% (4 of 34) of a cohort with early onset absence epilepsy (EOAE) had GLUT1 deficiency. This study uses a new cohort of 55 patients with EOAE to confirm that finding. Patients with typical absence seizures beginning before 4 years of age were screened for solute carrier family 2 (facilitated glucose transporter), member 1 (SLC2A1) mutations or deletions. All had generalized spike-waves on electroencephalography (EEG). Those with tonic and/or atonic seizures were excluded. Mutations were found in 7 (13%) of 55 cases, including five missense mutations, an in-frame deletion leading to loss of a single amino acid, and a deletion spanning two exons. Over both studies, 11 (12%) of 89 probands with EOAE have GLUT1 deficiency. Given the major treatment and genetic counseling implications, this study confirms that SLC2A1 mutational analysis should be strongly considered in EOAE.

Familial adult myoclonic epilepsy: recognition of mild phenotypes and refinement of the 2q locus.

BACKGROUND: Familial adult myoclonic epilepsy (FAME) is an autosomal dominant syndrome characterized by a core triad of cortical tremor, multifocal myoclonus, and generalized tonic-clonic seizures. OBJECTIVES: To expand the phenotypic spectrum of FAME, to highlight diagnostic pointers to this underrecognized disorder, and to refine the FAME2 genetic locus. DESIGN: Observational family study. SETTING: The study was coordinated in a tertiary academic hospital, with data acquired in diverse primary, secondary, and tertiary care settings. PARTICIPANTS: Consenting members of a single large family. RESULTS: A 6-generation FAME kindred of European descent was ascertained in New Zealand and Australia. Affected family members (N = 55) had fine hand tremor, with onset typically in adolescence (median age, 15 years; age range, 4-60 years). Proximal myoclonus was present in 44 of 55 (80%), arising later than hand tremor (median age, 17 years; age range, 5-60 years). Generalized tonic-clonic seizures occurred in 8 of 55 (15%), with a median age at onset of 43.5 years (age range, 18-76 years). Neurophysiological testing confirmed features of cortical reflex myoclonus. Genetic mapping narrows the FAME2 (OMIM 607876) locus on chromosome 2 to a 13.3-megabase interval, harboring 99 known protein-coding genes. CONCLUSIONS: The most common FAME phenotype in this large family is mild postural hand tremor resembling essential tremor, combined with subtle proximal myoclonus. Generalized tonic-clonic seizures are uncommon and occur around sleep onset following severe generalized myoclonus.

Glucose transporter 1 deficiency in the idiopathic generalized epilepsies.

OBJECTIVE: We examined whether glucose transporter 1 (GLUT1) deficiency causes common idiopathic generalized epilepsies (IGEs). METHODS: The IGEs are common, heritable epilepsies that usually follow complex inheritance; currently little is known about their genetic architecture. Previously considered rare, GLUT1 deficiency, due to mutations in SLC2A1, leads to failure of glucose transport across the blood-brain barrier and inadequate glucose for brain metabolism. GLUT1 deficiency was first associated with an encephalopathy and more recently found in rare dominant families with epilepsy and paroxysmal exertional dyskinesia (PED). Five hundred four probands with IGEs and 470 controls underwent SLC2A1 sequencing. Glucose transport was assayed following expression of SLC2A1 variants in Xenopus oocytes. All available relatives were phenotyped, and SLC2A1 was sequenced. RESULTS: Functionally validated mutations in SLC2A1 were present in 7 of 504 (1.4%) probands and 0 of 470 controls. PED, undiagnosed prior to study, occurred in 1 proband and 3 of 13 relatives with mutations. The IGEs in probands and relatives were indistinguishable from typical IGE. Three cases (0.6%) had mutations of large functional effect and showed autosomal dominant inheritance or were de novo. Four (0.8%) cases had a subtle functional effect; 2 showed possible dominant inheritance, and 2 did not. These alleles leading to subtle functional impairment may contribute to complex, polygenic inheritance of IGE. INTERPRETATION: SLC2A1 mutations contribute to approximately 1% of IGE both as a dominant gene and as a susceptibility allele in complex inheritance. Diagnosis of GLUT1 deficiency has important treatment (ketogenic diet) and genetic counseling implications. The mechanism of restricted glucose delivery differs from the current focus on IGEs as ion channel disorders.

Timing of de novo mutagenesis--a twin study of sodium-channel mutations.

De novo mutations are a cause of sporadic disease, but little is known about the developmental timing of such mutations. We studied concordant and discordant monozygous twins with de novo mutations in the sodium channel α1 subunit gene (SCN1A) causing Dravet's syndrome, a severe epileptic encephalopathy. On the basis of our findings and the literature on mosaic cases, we conclude that de novo mutations in SCN1A may occur at any time, from the premorula stage of the embryo (causing disease in the subject) to adulthood (with mutations in the germ-line cells of parents causing disease in offspring).

Familial mesial temporal lobe epilepsy: a benign epilepsy syndrome showing complex inheritance.

Temporal lobe epilepsy is the commonest partial epilepsy of adulthood. Although generally perceived as an acquired disorder, several forms of familial temporal lobe epilepsy, with mesial or lateral seizure semiology, have been described. Descriptions of familial mesial temporal lobe epilepsy have varied widely from a benign epilepsy syndrome with prominent déjà vu and without antecedent febrile seizures or magnetic resonance imaging abnormalities, to heterogeneous, but generally more refractory epilepsies, often with a history of febrile seizures and with frequent hippocampal atrophy and high T2 signal on magnetic resonance imaging. Compelling evidence of a genetic aetiology (rather than chance aggregation) in familial mesial temporal lobe epilepsy has come from twin studies. Dominant inheritance has been reported in two large families, though the usual mode of inheritance is not known. Here, we describe clinical and neurophysiological features of 20 new mesial temporal lobe epilepsy families including 51 affected individuals. The epilepsies in these families were generally benign, and febrile seizure history was infrequent (9.8%). No evidence of hippocampal sclerosis or dysplasia was present on brain imaging. A single individual underwent anterior temporal lobectomy, with subsequent seizure freedom and histopathological evidence of hippocampal sclerosis was not found. Inheritance patterns in probands' relatives were analysed in these families, together with 19 other temporal lobe epilepsy families previously reported by us. Observed frequencies of epilepsies in relatives were lower than predicted by dominant Mendelian models, while only a minority (8/39) of families could be compatible with recessive inheritance. These findings strongly suggest that complex inheritance, similar to that widely accepted in the idiopathic generalized epilepsies, is the usual mode of inheritance in familial mesial temporal lobe epilepsy. This disorder, which appears to be relatively common, and not typically associated with hippocampal sclerosis, is an appropriate target for contemporary approaches to complex disorders such as genome-wide association studies for common genetic variants or deep sequencing for rare variants.

Familial Lennox-Gastaut syndrome in male siblings with a novel DCX mutation and anterior pachygyria.

Lennox-Gastaut syndrome (LGS) has numerous causes,but only rarely has familial recurrence been observed. We studied a family in which three male members had severe epilepsy and intellectual disability. The proband had seizure onset at 7 years of age with atonic, myoclonic, atypical absence, and tonic seizures with slow spike-wave on electroencephalography (EEG). One living sibling had a similar clinical pattern. One deceased sibling was known to have had seizures with intellectual disability. Neuroimaging revealed anterior predominant pachygyria. DNA sequencing of the gene doublecortin (DCX) on the X chromosome revealed a novel missense mutation in the two living affected male siblings. The occurrence of three affected male family members with proven or suspected LGS in this family was puzzling and only solved by a combination of magnetic resonance (MR) and molecular genetics evaluations. This finding provided essential information for genetic counseling.

Reduced variance in monozygous twins for multiple MR parameters: implications for disease studies and the genetic basis of brain structure.

Twin studies offer the opportunity to determine the relative contribution of genes versus environment in traits of interest. Here, we investigate the extent to which variance in brain structure is reduced in monozygous twins with identical genetic make-up. We investigate whether using twins as compared to a control population reduces variability in a number of common magnetic resonance (MR) structural measures, and we investigate the location of areas under major genetic influences. This is fundamental to understanding the benefit of using twins in studies where structure is the phenotype of interest. Twenty-three pairs of healthy MZ twins were compared to matched control pairs. Volume, T2 and diffusion MR imaging were performed as well as spectroscopy (MRS). Images were compared using (i) global measures of standard deviation and effect size, (ii) voxel-based analysis of similarity and (iii) intra-pair correlation. Global measures indicated a consistent increase in structural similarity in twins. The voxel-based and correlation analyses indicated a widespread pattern of increased similarity in twin pairs, particularly in frontal and temporal regions. The areas of increased similarity were most widespread for the diffusion trace and least widespread for T2. MRS showed consistent reduction in metabolite variation that was significant in the temporal lobe N-acetylaspartate (NAA). This study has shown the distribution and magnitude of reduced variability in brain volume, diffusion, T2 and metabolites in twins. The data suggest that evaluation of twins discordant for disease is indeed a valid way to attribute genetic or environmental influences to observed abnormalities in patients since evidence is provided for the underlying assumption of decreased variability in twins.

Balance impairment in chronic antiepileptic drug users: a twin and sibling study.

PURPOSE: Patients taking antiepileptic drugs (AEDs) have an increased incidence of fractures. This study investigated chronic AED use and physical contributors to falls risk using an AED-discordant, twin and sibling matched-pair approach, and assessed clinically relevant subgroups: AED polytherapy; longer-duration AED; and falls history. METHODS: Twenty-nine same-sex (mean age 44.9 years, 59% female), ambulatory, community-dwelling twin and sibling pairs, discordant for AED exposure (and AED-indication), were recruited. Validated clinical and laboratory tests of strength, gait, and balance were performed. Relevant AED levels, and fasting serum samples for 25-hydroxyvitamin D (25OHD), 1,25-dihydroxyvitamin D [1,25(OH)(2)D], and immunoreactive parathyroid hormone (iPTH) levels were taken. RESULTS: There were significant mean within-pair differences in tests of static and dynamic balance, with the AED user having poorer balance function than the AED nonuser. No difference was seen in lower limb strength or gait measures. Increased duration of AED therapy and AED polytherapy were independent predictors of increased sway. No significant within-pair differences were seen in fasting serum levels of 1,25(OH)(2)D, 25OHD and iPTH after Bonferroni correction. DISCUSSION: Balance performance is impaired in AED users compared to their matched nonuser siblings. Pairs where the AED users took AED polytherapy, or had a longer duration of AED use, had more impaired balance performance. These balance deficits may contribute to the increased rate of fractures in this population.

Obstetric events as a risk factor for febrile seizures: a community-based twin study.

Adverse events during the perinatal period have traditionally been thought to contribute to the risk of febrile seizures although an association has not been found in large epidemiological studies. Disease-discordant twins provide a means to assess the role of non-shared environmental factors while matching for confounding factors and avoiding difficulties of epidemiological studies in singletons. This study aimed to examine the association of obstetric events and febrile seizures in a community-based twin study. Twenty-one twin pairs discordant for febrile seizures were ascertained from a community-based twin register. Obstetric events were scored using the McNeil-Sjöström Scale for Obstetric Complications and expressed as a summary score (OC score). The frequency of individual obstetric events in affected and unaffected twins, the within-pair differences in OC scores and other markers of perinatal risk including birthweight, birth order and Apgar scores were examined. No significant difference was found in the frequency of individual obstetric events, nor in OC scores between affected and unaffected twins. No differences in birth weight, birth order, 1- or 5-minute Apgar scores were observed. Our results confirm previous findings that obstetric events are not associated with the risk of febrile seizures.

Gene expression analysis in absence epilepsy using a monozygotic twin design.

PURPOSE: To identify genes involved in idiopathic absence epilepsies by analyzing gene expression using a monozygotic (MZ) twin design. METHODS: Genome-wide gene expression in lymphoblastoid cell lines (LCLs) was determined using microarrays derived from five discordant and four concordant MZ twin pairs with idiopathic absence epilepsies and five unaffected MZ twin pairs. Gene expression was analyzed using three strategies: discordant MZ twins were compared as matched pairs, MZ twins concordant for epilepsy were compared to control MZ twins, and a singleton design of affected versus unaffected MZ twin individuals was used irrespective of twin pairing. An overlapping gene list was generated from these analyses. Dysregulation of genes recognized from the microarray experiment was validated using quantitative real time PCR (qRT-PCR) in the twin sample and in an independent sample of 18 sporadic absence cases and 24 healthy controls. RESULTS: Sixty-five probe sets were identified from the three combined microarray analysis strategies. Sixteen genes were chosen for validation and nine of these genes confirmed by qRT-PCR in the twin sample. Differential expression for EGR1 (an immediate early gene) and RCN2 (coding for the calcium-binding protein Reticulocalbin 2) were reconfirmed by qRT-PCR in the independent sample. DISCUSSION: Using a unique sample of discordant MZ twins, our study identified genes with altered expression, which suggests novel mechanisms in idiopathic absence epilepsy. Dysregulation of EGR1 and RCN2 is implicated in idiopathic absence epilepsy.