Mutations in the mammalian target of rapamycin pathway regulators NPRL2 and NPRL3 cause focal epilepsy.

OBJECTIVE: Focal epilepsies are the most common form observed and have not generally been considered to be genetic in origin. Recently, we identified mutations in DEPDC5 as a cause of familial focal epilepsy. In this study, we investigated whether mutations in the mammalian target of rapamycin (mTOR) regulators, NPRL2 and NPRL3, also contribute to cases of focal epilepsy. METHODS: We used targeted capture and next-generation sequencing to analyze 404 unrelated probands with focal epilepsy. We performed exome sequencing on two families with multiple members affected with focal epilepsy and linkage analysis on one of these. RESULTS: In our cohort of 404 unrelated focal epilepsy patients, we identified five mutations in NPRL2 and five in NPRL3. Exome sequencing analysis of two families with focal epilepsy identified NPRL2 and NPRL3 as the top candidate-causative genes. Some patients had focal epilepsy associated with brain malformations. We also identified 18 new mutations in DEPDC5. INTERPRETATION: We have identified NPRL2 and NPRL3 as two new focal epilepsy genes that also play a role in the mTOR-signaling pathway. Our findings show that mutations in GATOR1 complex genes are the most significant cause of familial focal epilepsy identified to date, including cases with brain malformations. It is possible that deregulation of cellular growth control plays a more important role in epilepsy than is currently recognized.

Mutations in KCNT1 cause a spectrum of focal epilepsies.

Autosomal dominant mutations in the sodium-gated potassium channel subunit gene KCNT1 have been associated with two distinct seizure syndromes, nocturnal frontal lobe epilepsy (NFLE) and malignant migrating focal seizures of infancy (MMFSI). To further explore the phenotypic spectrum associated with KCNT1, we examined individuals affected with focal epilepsy or an epileptic encephalopathy for mutations in the gene. We identified KCNT1 mutations in 12 previously unreported patients with focal epilepsy, multifocal epilepsy, cardiac arrhythmia, and in a family with sudden unexpected death in epilepsy (SUDEP), in addition to patients with NFLE and MMFSI. In contrast to the 100% penetrance so far reported for KCNT1 mutations, we observed incomplete penetrance. It is notable that we report that the one KCNT1 mutation, p.Arg398Gln, can lead to either of the two distinct phenotypes, ADNFLE or MMFSI, even within the same family. This indicates that genotype-phenotype relationships for KCNT1 mutations are not straightforward. We demonstrate that KCNT1 mutations are highly pleiotropic and are associated with phenotypes other than ADNFLE and MMFSI. KCNT1 mutations are now associated with Ohtahara syndrome, MMFSI, and nocturnal focal epilepsy. They may also be associated with multifocal epilepsy and cardiac disturbances.

A mutation in the Golgi Qb-SNARE gene GOSR2 causes progressive myoclonus epilepsy with early ataxia.

The progressive myoclonus epilepsies (PMEs) are a group of predominantly recessive disorders that present with action myoclonus, tonic-clonic seizures, and progressive neurological decline. Many PMEs have similar clinical presentations yet are genetically heterogeneous, making accurate diagnosis difficult. A locus for PME was mapped in a consanguineous family with a single affected individual to chromosome 17q21. An identical-by-descent, homozygous mutation in GOSR2 (c.430G>T, p.Gly144Trp), a Golgi vesicle transport gene, was identified in this patient and in four apparently unrelated individuals. A comparison of the phenotypes in these patients defined a clinically distinct PME syndrome characterized by early-onset ataxia, action myoclonus by age 6, scoliosis, and mildly elevated serum creatine kinase. This p.Gly144Trp mutation is equivalent to a loss of function and results in failure of GOSR2 protein to localize to the cis-Golgi.

'North Sea' progressive myoclonus epilepsy: phenotype of subjects with GOSR2 mutation.

We previously identified a homozygous mutation in the Golgi SNAP receptor complex 2 gene (GOSR2) in six patients with progressive myoclonus epilepsy. To define the syndrome better we analysed the clinical and electrophysiological phenotype in 12 patients with GOSR2 mutations, including six new unrelated subjects. Clinical presentation was remarkably similar with early onset ataxia (average 2 years of age), followed by myoclonic seizures at the average age of 6.5 years. Patients developed multiple seizure types, including generalized tonic clonic seizures, absence seizures and drop attacks. All patients developed scoliosis by adolescence, making this an important diagnostic clue. Additional skeletal deformities were present, including pes cavus in four patients and syndactyly in two patients. All patients had elevated serum creatine kinase levels (median 734 IU) in the context of normal muscle biopsies. Electroencephalography revealed pronounced generalized spike and wave discharges with a posterior predominance and photosensitivity in all patients, with focal EEG features seen in seven patients. The disease course showed a relentless decline; patients uniformly became wheelchair bound (mean age 13 years) and four had died during their third or early fourth decade. All 12 cases had the same variant (c.430G>T, G144W) and haplotype analyses confirmed a founder effect. The cases all came from countries bounding the North Sea, extending to the coastal region of Northern Norway. 'North Sea' progressive myoclonus epilepsy has a homogeneous clinical presentation and relentless disease course allowing ready identification from the other progressive myoclonus epilepsies.

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

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

Array-based gene discovery with three unrelated subjects shows SCARB2/LIMP-2 deficiency causes myoclonus epilepsy and glomerulosclerosis.

Action myoclonus-renal failure syndrome (AMRF) is an autosomal-recessive disorder with the remarkable combination of focal glomerulosclerosis, frequently with glomerular collapse, and progressive myoclonus epilepsy associated with storage material in the brain. Here, we employed a novel combination of molecular strategies to find the responsible gene and show its effects in an animal model. Utilizing only three unrelated affected individuals and their relatives, we used homozygosity mapping with single-nucleotide polymorphism chips to localize AMRF. We then used microarray-expression analysis to prioritize candidates prior to sequencing. The disorder was mapped to 4q13-21, and microarray-expression analysis identified SCARB2/Limp2, which encodes a lysosomal-membrane protein, as the likely candidate. Mutations in SCARB2/Limp2 were found in all three families used for mapping and subsequently confirmed in two other unrelated AMRF families. The mutations were associated with lack of SCARB2 protein. Reanalysis of an existing Limp2 knockout mouse showed intracellular inclusions in cerebral and cerebellar cortex, and the kidneys showed subtle glomerular changes. This study highlights that recessive genes can be identified with a very small number of subjects. The ancestral lysosomal-membrane protein SCARB2/LIMP-2 is responsible for AMRF. The heterogeneous pathology in the kidney and brain suggests that SCARB2/Limp2 has pleiotropic effects that may be relevant to understanding the pathogenesis of other forms of glomerulosclerosis or collapse and myoclonic epilepsies.

Investigation of the 15q13.3 CNV as a genetic modifier for familial epilepsies with variable phenotypes.

Incomplete penetrance and variable phenotypic expression are characteristic of a number of syndromes of familial epilepsy. The purpose of the present investigation is to determine if the 15q13.3 copy number deletion functions as a locus modifying the epilepsy phenotype caused by other known or presumed pathogenic mutations segregating in families with epilepsies. No 15q13.3 microdeletions were detected in 756 affected or definite obligate carrier individuals across 151 families selected on the basis of having multiple members affected with epilepsy and showing a range of seizure types. Therefore, the 15q13.3 microdeletion does not act as a genetic modifier in this cohort of families and is not responsible for any of the genetic heterogeneity hypothesized to account for failure to detect linkage in previous genome-wide scans in five of the larger families included in this study.

Epilepsy and mental retardation limited to females with PCDH19 mutations can present de novo or in single generation families.

BACKGROUND: Epilepsy and mental retardation limited to females (EFMR) is an intriguing X-linked disorder affecting heterozygous females and sparing hemizygous males. Mutations in the protocadherin 19 (PCDH19) gene have been identified in seven unrelated families with EFMR. METHODS AND RESULTS: Here, we assessed the frequency of PCDH19 mutations in individuals with clinical features which overlap those of EFMR. We analysed 185 females from three cohorts: 42 with Rett syndrome who were negative for MECP2 and CDKL5 mutations, 57 with autism spectrum disorders, and 86 with epilepsy with or without intellectual disability. No mutations were identified in the Rett syndrome and autism spectrum disorders cohorts suggesting that despite sharing similar clinical characteristics with EFMR, PCDH19 mutations are not generally associated with these disorders. Among the 86 females with epilepsy (of whom 51 had seizure onset before 3 years), with or without intellectual disability, we identified two (2.3%) missense changes. One (c.1671C-->G, p.N557K), reported previously without clinical data, was found in two affected sisters, the first EFMR family without a multigenerational family history of affected females. The second, reported here, is a novel de novo missense change identified in a sporadic female. The change, p.S276P, is predicted to result in functional disturbance of PCDH19 as it affects a highly conserved residue adjacent to the adhesion interface of EC3 of PCDH19. CONCLUSIONS: This de novo PCDH19 mutation in a sporadic female highlights that mutational analysis should be considered in isolated instances of girls with infantile onset seizures and developmental delay, in addition to those with the characteristic family history of EFMR.

Epilepsy and mental retardation limited to females: an under-recognized disorder.

Epilepsy and Mental Retardation limited to Females (EFMR) which links to Xq22 has been reported in only one family. We aimed to determine if there was a distinctive phenotype that would enhance recognition of this disorder. We ascertained four unrelated families (two Australian, two Israeli) where seizures in females were transmitted through carrier males. Detailed clinical assessment was performed on 58 individuals, using a validated seizure questionnaire, neurological examination and review of EEG and imaging studies. Gene localization was examined using Xq22 microsatellite markers. Twenty-seven affected females had a mean seizure onset of 14 months (range 6-36) typically presenting with convulsions. All had convulsive attacks at some stage, associated with fever in 17 out of 27 (63%). Multiple seizure types occurred including tonic-clonic (26), tonic (4), partial (11), absence (5), atonic (3) and myoclonic (4). Seizures ceased at mean 12 years. Developmental progress varied from normal (7), to always delayed (4) to normal followed by regression (12). Intellect ranged from normal to severe intellectual disability (ID), with 67% of females having ID or being of borderline intellect. Autistic (6), obsessive (9) and aggressive (7) features were prominent. EEGs showed generalized and focal epileptiform abnormalities. Five obligate male carriers had obsessional tendencies. Linkage to Xq22 was confirmed (maximum lod 3.5 at = 0). We conclude that EFMR is a distinctive, under-recognized familial syndrome where girls present with convulsions in infancy, often associated with intellectual impairment and autistic features. The unique inheritance pattern with transmission by males is perplexing. Clinical recognition is straightforward in multiplex families due to the unique inheritance pattern; however, this disorder should be considered in smaller families where females alone have seizures beginning in infancy, particularly in the setting of developmental delay. In single cases, diagnosis will depend on identification of the molecular basis.

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.

A recurrent de novo mutation in KCNC1 causes progressive myoclonus epilepsy.

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.

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

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

Abnormal Processing of Autophagosomes in Transformed B Lymphocytes from SCARB2-Deficient Subjects.

Mutations of the intrinsic lysosomal membrane protein SCARB2 cause action myoclonus-renal failure syndrome (AMRF syndrome), a rare disease characterized by renal and neurological manifestations. In this study, examination of Cos7 cells transfected with SCARB2 cDNA derived from two patients with AMRF syndrome showed that the resultant protein was truncated and was not incorporated into vesicular structures, as occurred with full-length SCARB2 cDNA. Mutant SCARB2 protein failed to colocalize with lysosomes and was found in the endoplasmic reticulum or the cytosol indicating a loss of function. Cultured skin fibroblast and Epstein-Barr virus-transformed lymphoblastoid B cell lines (LCLs) were created from these two patients. Despite the loss of SCARB2 function, studies with lysosomal-associated membrane protein (LAMP) 1 and LAMP2 demonstrated normal lysosomal numbers in fibroblasts and LCLs. Immunofluorescence microscopy using anti-LAMP1 and anti-LAMP2 antibodies also showed normal lysosomal structures in fibroblasts. There was no change in the morphology of fibroblasts examined by electron microscopy compared with cells from unaffected individuals. By contrast, LCLs from individuals bearing SCARB2 mutations had large intracellular vesicles that resembled autophagosomes and contained heterogeneous cellular debris. Some of the autophagosomes were seen to be extruding cellular contents into the media. Furthermore, LCLs had elevated levels of microtubule-associated protein light chain 3-II, consistent with increased autophagy. These data demonstrate that SCARB2 mutations are associated with an inability to process autophagosomes in B lymphocytes, suggesting a novel function for SCARB2 in immune function.

Mutation of SCARB2 in a patient with progressive myoclonus epilepsy and demyelinating peripheral neuropathy.

OBJECTIVE: To report the detection of mutations in the SCARB2 gene in a previously described patient with progressive myoclonus epilepsy (PME) and demyelinating peripheral neuropathy. DESIGN: Case report. SETTING: Epilepsy Genetics Research Laboratory and Epilepsy Service in a tertiary care center. PATIENT: A 27-year old male patient with PME with preserved intellect and peripheral neuropathy. RESULTS: We have solved a previously reported case of PME, preserved intellect, and demyelinating peripheral neuropathy. The patient is a compound heterozygote for 2 mutations in the SCARB2 gene, which has recently been found to be a cause of PME. CONCLUSIONS: Demyelinating neuropathy is a clinical clue to the presence of SCARB2 mutations in PME.

X-linked protocadherin 19 mutations cause female-limited epilepsy and cognitive impairment.

Epilepsy and mental retardation limited to females (EFMR) is a disorder with an X-linked mode of inheritance and an unusual expression pattern. Disorders arising from mutations on the X chromosome are typically characterized by affected males and unaffected carrier females. In contrast, EFMR spares transmitting males and affects only carrier females. Aided by systematic resequencing of 737 X chromosome genes, we identified different protocadherin 19 (PCDH19) gene mutations in seven families with EFMR. Five mutations resulted in the introduction of a premature termination codon. Study of two of these demonstrated nonsense-mediated decay of PCDH19 mRNA. The two missense mutations were predicted to affect adhesiveness of PCDH19 through impaired calcium binding. PCDH19 is expressed in developing brains of human and mouse and is the first member of the cadherin superfamily to be directly implicated in epilepsy or mental retardation.