[Abstract unavailable

Developmental and epileptic encephalopathy (DEE) is a group of conditions characterized by the co-occurrence of epilepsy and intellectual disability (ID), typically with developmental plateauing or regression associated with frequent epileptiform activity. The cause of DEE remains unknown in the majority of cases. We performed whole-genome sequencing (WGS) in 197 individuals with unexplained DEE and pharmaco-resistant seizures and in their unaffected parents. We focused our attention on de novo mutations (DNMs) and identified candidate genes containing such variants. We sought to identify additional subjects with DNMs in these genes by performing targeted sequencing in another series of individuals with DEE and by mining various sequencing datasets. We also performed meta-analyses to document enrichment of DNMs in candidate genes by leveraging our WGS dataset with those of several DEE and ID series. By combining these strategies, we were able to provide a causal link between DEE and the following genes: NTRK2, GABRB2, CLTC, DHDDS, NUS1, RAB11A, GABBR2, and SNAP25. Overall, we established a molecular diagnosis in 63/197 (32%) individuals in our WGS series. The main cause of DEE in these individuals was de novo point mutations (53/63 solved cases), followed by inherited mutations (6/63 solved cases) and de novo CNVs (4/63 solved cases). De novo missense variants explained a larger proportion of individuals in our series than in other series that were primarily ascertained because of ID. Moreover, these DNMs were more frequently recurrent than those identified in ID series. These observations indicate that the genetic landscape of DEE might be different from that of ID without epilepsy.

Lafora's disease (LD; OMIM 254780) is an autosomal recessive form of progressive myoclonus epilepsy characterized by seizures and cumulative neurological deterioration. Onset occurs during late childhood and usually results in death within ten years of the first symptoms. With few exceptions, patients follow a homogeneous clinical course despite the existence of genetic heterogeneity. Biopsy of various tissues, including brain, revealed characteristic polyglucosan inclusions called Lafora bodies, which suggested LD might be a generalized storage disease. Using a positional cloning approach, we have identified at chromosome 6q24 a novel gene, EPM2A, that encodes a protein with consensus amino acid sequence indicative of a protein tyrosine phosphatase (PTP). mRNA transcripts representing alternatively spliced forms of EPM2A were found in every tissue examined, including brain. Six distinct DNA sequence variations in EPM2A in nine families, and one homozygous microdeletion in another family, have been found to cosegregate with LD. These mutations are predicted to cause deleterious effects in the putative protein product, named laforin, resulting in LD.

Rett syndrome is caused by mutations in the gene MECP2 in ∼80% of affected individuals. We describe a previously unknown MeCP2 isoform. Mutations unique to this isoform and the absence, until now, of identified mutations specific to the previously recognized protein indicate an important role for the newly discovered molecule in the pathogenesis of Rett syndrome.Rett syndrome (RTT; OMIM 312750) is characterized by onset, in girls, of a gradual slowing of neurodevelopment in the second half of the first year of life that proceeds towards stagnation by age 4 years, followed by regression and loss of acquired fine motor and communication skills. A pseudostationary period follows during which a picture of preserved ambulation, aberrant communication and stereotypic hand wringing approximates early autism. Regression, however, remains insidiously ongoing and ultimately results in profound mental retardation 1 .Up to 80% of individuals with RTT have mutations 2,3 in exons 3 and 4 of the four-exon gene MECP2 (Fig. 1a) 4 encoding the transcriptional repressor MeCP2. In the known transcript of the gene, all four exons are used, the translation start site is in exon 2, and exon 1 and most of exon 2 form the 5′ untranslated region (UTR) 4 . For clarity, we refer to this transcript as MECP2A and its encoded protein as MeCP2A. We sought to identify MECP2 splice variants contributing new coding sequence that might contain mutations in the remaining individuals with RTT. Inspection of the 5′ UTR showed that, whereas exon 2 has a number of in-frame stop codons upstream of the ATG start codon, exon 1 contains an open reading frame across its entire length, including an ATG. Submitting a theoretical construct composed of exons 1, 3 and 4 to the ATGpr program (http://www.hri.co.jp/atgpr/), which predicts the likelihood that an ATG will be an initiation codon based on the significance of its surrounding Kozak nucleotide context, returned a reliability score of 97%, as compared with 64% for MECP2A. A search in EST databases identified eight examples of our theorized transcript, which we named MECP2B (Fig. 1b), as compared with 14 examples of MECP2A. MECP2B is predicted to encode a new isoform, MeCP2B, with an alternative, longer N terminus determined by exon 1 (see Supplementary Table 1 online).To confirm that MECP2B is expressed and not merely an artifact of cDNA library preparation, we amplified cDNA by PCR from a variety of tissues using a 5′ primer in exon 1 and a 3′ primer in exon 3 (Fig. 1a). We obtained two PCR products corresponding in size and sequence to MECP2A and MECP2B in all tissues, including fetal and adult brain and different brain subregions (Fig. 1c). Results in mouse were similar (Fig. 1c). We quantified the expression levels of the two transcripts in adult human brain. Expression of MECP2B was ten times higher than that of MECP2A (Fig. 1d). We studied the subcellular localization of MeCP2B after transfection of 3′ myc-tagged MECP2B into COS-7 cells and found it to be principally in the nucleus (Fig. 1e).To deter...

Angelman syndrome (AS) is a severe neurodevelopmental disorder resulting from a deletion/mutation in maternal chromosome 15q11–13. The genes in 15q11–13 contributing to the full array of the clinical phenotype are not fully identified. This study examines whether a loss or reduction in the GABAAreceptor β3subunit (GABRB3) gene, contained within the AS deletion region, may contribute to the overall severity of AS. Disrupting the gabrb3 gene in mice produces electroencephalographic abnormalities, seizures, and behavior that parallel those seen in AS. The seizures that are observed in these mice showed a pharmacological response profile to antiepileptic medications similar to that observed in AS. Additionally, these mice exhibited learning and memory deficits, poor motor skills on a repetitive task, hyperactivity, and a disturbed rest–activity cycle, features all common to AS. The loss of the single gene, gabrb3, in these mice is sufficient to cause phenotypic traits that have marked similarities to the clinical features of AS, indicating that impaired expression of the GABRB3 gene in humans probably contributes to the overall phenotype of Angelman syndrome. At least one other gene, the E6-associated protein ubiquitin-protein ligase (UBE3A) gene, has been implicated in AS, so the relative contribution of the GABRB3 gene alone or in combination with other genes remains to be established.

Lafora disease is a progressive myoclonus epilepsy with onset typically in the second decade of life and death within 10 years. Lafora bodies, deposits of abnormally branched, insoluble glycogen-like polymers, form in neurons, muscle, liver, and other tissues. Approximately half of the cases of Lafora disease result from mutations in the EPM2A gene, which encodes laforin, a member of the dual-specificity protein phosphatase family that additionally contains a glycogen binding domain. The molecular basis for the formation of Lafora bodies is completely unknown. Glycogen, a branched polymer of glucose, contains a small amount of covalently linked phosphate whose origin and function are obscure. We report here that recombinant laforin is able to release this phosphate in vitro, in a time-dependent reaction with an apparent Km for glycogen of 4.5 mg/ml. Mutations of laforin that disable the glycogen binding domain also eliminate its ability to dephosphorylate glycogen. We have also analyzed glycogen from a mouse model of Lafora disease, Epm2a ؊/؊ mice, which develop Lafora bodies in several tissues. Glycogen isolated from these mice had a 40% increase in the covalent phosphate content in liver and a 4-fold elevation in muscle. We propose that excessive phosphorylation of glycogen leads to aberrant branching and Lafora body formation. This study provides a molecular link between an observed biochemical property of laforin and the phenotype of a mouse model of Lafora disease. The results also have important implications for glycogen metabolism generally.polyglucosan ͉ Lafora bodies ͉ Epm2a ͉ phosphate

Lafora disease is a progressive myoclonus epilepsy with onset in the teenage years followed by neurodegeneration and death within 10 years. A characteristic is the widespread formation of poorly branched, insoluble glycogen-like polymers (polyglucosan) known as Lafora bodies, which accumulate in neurons, muscle, liver, and other tissues. Approximately half of the cases of Lafora disease result from mutations in the EPM2A gene, which encodes laforin, a member of the dual specificity protein phosphatase family that is able to release the small amount of covalent phosphate normally present in glycogen. In studies of Epm2a ؊/؊ mice that lack laforin, we observed a progressive change in the properties and structure of glycogen that paralleled the formation of Lafora bodies. At three months, glycogen metabolism remained essentially normal, even though the phosphorylation of glycogen has increased 4-fold and causes altered physical properties of the polysaccharide. By 9 months, the glycogen has overaccumulated by 3-fold, has become somewhat more phosphorylated, but, more notably, is now poorly branched, is insoluble in water, and has acquired an abnormal morphology visible by electron microscopy. These glycogen molecules have a tendency to aggregate and can be recovered in the pellet after low speed centrifugation of tissue extracts. The aggregation requires the phosphorylation of glycogen. The aggregrated glycogen sequesters glycogen synthase but not other glycogen metabolizing enzymes. We propose that laforin functions to suppress excessive glycogen phosphorylation and is an essential component of the metabolism of normally structured glycogen.Glycogen is a branched polymer of glucose that acts as a repository of energy used in times of need (1). Liver and skeletal muscle contain the two largest deposits in mammals, but heart, brain, adipose, as well as many other tissues synthesize the polymer. Polymerization occurs via ␣-1,4-glycosidic linkages between glucose residues, with branch points introduced by ␣-1,6-glycosidic linkages. The frequency of branching determines the topology of glycogen and distinguishes it from the carbohydrate moiety of starch (2). A unique three-dimensional structure of glycogen cannot be determined experimentally because of the polydispersity of the molecule, but a widely accepted model of its structure has been proposed (3). Glycogen is composed of successive layers, or tiers, of glucose residues; a full size molecule consists of 12 tiers, with M r ϭ ϳ10 7 and a diameter of ϳ40 nm (4). Glycogen has been reported to contain small amounts of covalently linked phosphate, on the order of 0.064% by weight or 0.121% mol/mol (5-7). The mechanism by which the phosphate is introduced remains unclear. One suggestion (5) has been the existence of a glucose-1-phosphate transferase that would form C1-C6 bridging phosphodiesters, but this enzyme has not been further defined at the molecular level. In plant amylopectin, C1 and C3 phosphomonoesters have been shown to be formed by dikinase enzymes (8), but...

Lafora progressive myoclonus epilepsy is characterized by pathognomonic endoplasmic reticulum (ER)-associated polyglucosan accumulations. We previously discovered that mutations in EPM2A cause Lafora disease. Here, we identify a second gene associated with this disease, NHLRC1 (also called EPM2B), which encodes malin, a putative E3 ubiquitin ligase with a RING finger domain and six NHL motifs. Laforin and malin colocalize to the ER, suggesting they operate in a related pathway protecting against polyglucosan accumulation and epilepsy.