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Lafora disease is the most severe teenage-onset progressive epilepsy, a unique form of glycogenosis with perikaryal accumulation of an abnormal form of glycogen, and a neurodegenerative disorder exhibiting an unusual generalized organellar disintegration. The disease is caused by mutations of the EPM2A gene, which encodes two isoforms of the laforin protein tyrosine phosphatase, having alternate carboxyl termini, one localized in the cytoplasm (endoplasmic reticulum) and the other in the nucleus. To date, all documented disease mutations, including the knockout mouse model deletion, have been in the segment of the protein common to both isoforms. It is therefore not known whether dysfunction of the cytoplasmic, nuclear, or both isoforms leads to the disease. In the present work, we identify six novel mutations, one of which, c.950insT (Q319fs), is the first mutation specific to the cytoplasmic laforin isoform, implicating this isoform in disease pathogenesis. To confirm this mutation's deleterious effect on laforin, we studied the resultant protein's subcellular localization and function and show a drastic reduction in its phosphatase activity, despite maintenance of its location at the endoplasmic reticulum.

Background: Lafora disease is a progressive myoclonus epilepsy with polyglucosan accumulations and a peculiar neurodegeneration with generalised organellar disintegration. It causes severe seizures, leading to dementia and eventually death in early adulthood. Methods: One Lafora disease gene, EPM2A , has been identified on chromosome 6q24. Locus heterogeneity led us to search for a second gene using a genome wide linkage scan in French-Canadian families. Results: We mapped a second Lafora disease locus, EPM2B , to a 2.2 Mb region at 6p22, a region known to code for several proteins, including kinesins. Kinesins are microtubule dependent motor proteins that are involved in transporting cellular components. In neurones, they play a major role in axonal and dendritic transport. Conclusion: Analysis of the present locus in other non- EPM2A families will reveal whether there is further locus heterogeneity. Identification of the disease gene will be of major importance towards our understanding of the pathogenesis of Lafora disease.

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.

We have identified an interacting partner protein (encoded by the human EPM2AIP1 gene (approved symbol)) for laforin, the product of the EPM2A gene, which is mutated in an autosomal recessive form of adolescent progressive myoclonus epilepsy. The EPM2AIP1 gene was identified in a screen for laforin-interacting proteins with a human brain cDNA library using the yeast two-hybrid system. The specificity of the interaction was confirmed by coimmunoprecipitation of in vivo-transfected protein and by using EPM2A deletion constructs. Subcellular colocalization of laforin and EPM2AIP1 protein was also demonstrated. The human EPM2AIP1 gene, corresponding to the KIAA0766 cDNA clone in the databases, was characterized and shown, like EPM2A, to be ubiquitously expressed. The gene, which comprises one large exon 1824 nucleotides in length and has alternative 3′ untranslated regions, maps to human chromosome 3p22.1. The function is currently not known and extensive analyses do not reveal any homology to other proteins or any obvious structural motifs. Because genetic heterogeneity in Lafora disease has been described, mutational analysis of the EPM2AIP1 gene was performed on non-EPM2A patients, but no mutations were found. The identification of this first binding partner for laforin promises to be an important step toward unraveling the underlying pathogenesis of this severest form of teenage-onset epilepsy.

DNA sequence and annotation of the entire human chromosome 7, encompassing nearly 158 million nucleotides of DNA and 1917 gene structures, are presented. To generate a higher order description, additional structural features such as imprinted genes, fragile sites, and segmental duplications were integrated at the level of the DNA sequence with medical genetic data, including 440 chromosome rearrangement breakpoints associated with disease. This approach enabled the discovery of candidate genes for developmental diseases including autism., link_to_OA_fulltext

Progressive myoclonus epilepsy of the Unverricht-Lundborg type (EPM1) is a recessively inherited neurodegenerative disease caused by loss-of-function mutations in the gene encoding cystatin B, a cysteine protease inhibitor. Mice with disruptions in this gene display myoclonic seizures, progressive ataxia, and cerebellar pathology closely paralleling EPM1 in humans. To provide further insight into our understanding of EPM1, we report pathological findings in brains from cystatin B-deficient mice. In addition to confirming the loss of cerebellar granular cell neurons by apoptosis, we identified additional neuronal apoptosis in young mutant mice (3–4 months old) within the hippocampal formation and entorhinal cortex. In older mutant mice (16–18 months old), there was also gliosis most marked in the presubiculum and parasubiculum of the hippocampal formation, as well as the entorhinal cortex, neocortex, and striatum. Furthermore, widespread white matter gliosis was also noted, which may be a secondary phenomenon. Within the cerebral cortex, cellular atrophy was a prominent finding in the superficial neurons of the prosubiculum. Finally, we show that mutant mice in either a “seizure-prone” or “seizure-resistant” genetic background display similar neuropathological changes. These findings indicate that neuronal atrophy is an important consequence of cystatin-B deficiency independent of seizure events, suggesting a physiological role for this protein in the maintenance of normal neuronal structure.

Phenytoin is an effective anticonvulsant, but high serum phenytoin concentrations may be associated with serious toxicity. The upper limit for the therapeutic serum concentration of phenytoin is considered to be 80 micromol/L. However, in some situations higher serum concentrations are needed to control seizures. The authors describe a 9-year-old girl who needed concentrations twice the normal amount to control recurrent episodes of decreased levels of consciousness. Except for nystagmus, she had no other signs of phenytoin toxicity. This patient highlights the critical principle in therapeutic drug monitoring of individualizing drug therapy. Although some patients receiving phenytoin may achieve seizure control with "subtherapeutic" levels (i.e.,40 micromol/L), others may need supratherapeutic levels, as was the case with this patient. Clinicians should be careful not to treat "numbers" (i.e., serum concentrations), but rather the patient's clinical condition, with a careful balance between therapeutic advantage and adverse effects.

A 19-year-old male patient presented with progressive myoclonic seizures and speech disorder. The patient had photosensitivity, a few episodes of sudden transient blindness, and infrequent complex visual auras, dysarthria and mild ataxia, frequent myoclonic jerks prominently in the legs and severe dementia. Microscopic examination of the axillary skin biopsy revealed periodic acid-Schiff positive inclusion bodies in abluminal side of the apocrine sweat gland acini. Molecular screening showed a homozygous R241X mutation in EPM2A. Genotyping helps in the correct diagnosis of the Lafora disease (LD), which may be difficult to diagnose based on the available histopathological testing only. Our study is an effort to determine the distribution of mutations in LD patients in our region.

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

First recognized by Andreas Rett in 1966 and rediscovered in 1983,1 Rett syndrome (RS) is among the most common causes of mental retardation, affecting upwards of 1 in 10,000 girls. Extensive clinical characterization has revealed a distinctive clinical entity. Stereotypic almost constant hand-rubbing in wakefulness is its most recognizable symptom. Social anxiety akin to autism is another. The latter increases the former, and the girls appear as though they are, and perhaps they truly are, wringing their hands in anxiety, hence the use of the term hand-wringing. The disease begins in mid-infancy with decelerating head growth after a period of normal development. The neurologic regression starts between 6 and 36 months with developmental arrest, and loss of or reduction in hand use, speech, and communication skills, and social interest. Additional features include a typical pattern of disease progression with the regression reaching a plateau and not progressing further, in contradistinction to other neurodegenerative diseases, and autonomic abnormalities including irregular breathing and cold blue extremities.2 RS is caused by mutations in the X-linked MECP2 gene, which encodes the …

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.

We compared epilepsy phenotypes with genotypes of Angelman syndrome (AS), including chromosome 15q11-13 deletions (class I), uniparental disomy (class II), methylation imprinting abnormalities (class III), and mutation in the UBE3A gene (class IV). Twenty patients were prospectively selected based on clinical cytogenetic and molecular diagnosis of AS. All patients had 6 to 72 hours of closed-circuit television videotaping and digitized electroencephalogrpahic (EEG) telemetry. Patients from all genotypic classes had characteristic EEGs with diffuse bifrontally dominant high-amplitude 1- to 3-Hz notched or triphasic or polyphasic slow waves, or slow and sharp waves. Class I patients had severe intractable epilepsy, most frequently with atypical absences and myoclonias and less frequently with generalized extensor tonic seizures or flexor spasms. Epileptic spasms were recorded in AS patients as old as 41 years. Aged-matched class II, III, and IV patients had either no epilepsy or drug-responsive mild epilepsy with relatively infrequent atypical absences, myoclonias, or atonic seizures. In conclusion, maternally inherited chromosome 15q11-13 deletions produce severe epilepsy. Loss-of-function UBE3A mutations, uniparental disomy, or methylation imprint abnormalities in AS are associated with relatively mild epilepsy. Involvement of other genes in the chromosome 15q11-13 deletion, such as GABRB3, may explain severe epilepsy in AS.

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2006

X-linked myopathy with excessive autophagy (XMEA) (MIM 310440) is an X-linked recessive boyhood-onset disorder of skeletal muscle with a slowly progressive course toward wasting of proximal muscles of the lower extremities and wheelchair dependency by the fifth decade of life. Involvement of distal leg muscles and upper extremities is clinically evident late in the disease.1–4 Skeletal muscle pathology is characterized by a constellation of rare and unique features. Autophagic vacuoles containing cellular debris are present in the sarcoplasm, and at the sarcolemma extruding their contents to the extracellular space. Myofibers containing vacuoles have multiple layers of basement membrane. Intense calcium accumulation is seen at the myofiber periphery, in the absence of necrosis. Extensive deposition of complement membrane attack complex (MAC) is present at the myofiber surface, without resultant cell lysis. Finally, class I major histocompatibility complex is expressed, despite the absence of inflammation.1–4 Nerve conduction studies (NCS) are normal but …

Angelman syndrome (AS) is a genetic disorder which produces severe mental retardation with concurrent epileptic seizures. AS typically results from a large deletion on maternal chromosome 15q11–13. The mutant mouse strain p cp (pink-eyed cleft palate) has a deletion in murine chromosome 7, syntenic to the human chromosomal region on 15 associated with AS, and may prove to be a valuable asset in addressing some of the pharmacological and behavioral consequences associated with AS. Changes in the density and properties of GABAA receptors in these mice is consistent with a loss of a GABAA gene cluster (subunits α5, β3, γ3) on murine chromosome 7. A large loss of inhibitory receptors could conceivably contribute to altered brain function possibly leading to seizure activity.

INTRODUCTION The intestine is a significant site of gluconeogenesis, with high expression of glucose 6 phosphatase. Abnormal forms of glycogen, including polyglucosan bodies, occur in the muscularis propria in a subset of patients with gastrointestinal motility disorders. Little is known, however, of the distribution of glycogen metabolic enzymes and related proteins in normal gastrointestinal tract, or effects of deficiencies of these proteins on the bowel. METHODS Key enzymes in glycogen metabolism, including glycogen synthase (GS), glycogen branching enzyme (GBE), malin and laforin were examined in stomach, ileum and colon of two mouse strains and in normal human bowel using immunohistochemistry with semiquantitiative analysis of epithelium, muscle, submucosal and myenteric plexus, validated by western blotting and positive and negative controls. The pathological effects of GBE, laforin and malin gene knockout on mouse gastrointestinal tract were also studied. RESULTS GS was present at high levels in the smooth muscle of muscularis propria, GBE in the majority of large ganglion cells of the submucosal and myenteric plexuses, and laforin and malin in submucosal ganglia. Analysis of human bowel showed no significant regional differences in the distribution of GBE, laforin and malin, GS was more prominent in the muscularis propria of the small intestine, compared with stomach and colon. GBE knockout produced a progressive severe myopathy with numerous polyglucosan bodies and vacuolation. Polyglucosan bodies were also present in both laforin and malin knockout models, but with a less severe phenotype than that seen in the GBE knockout, and were not seen in controls. CONCLUSION The distribution of glycogen metabolic proteins in the bowel wall of mouse and man shows a specific neuromuscular distribution for each pathway component. Defects in glycogen metabolism in mouse knockout models reproduce histological features of polyglucosan body myopathy in human digestive motility disorders.

Lafora disease (EPM2A and EPM2B genes); myoclonus epilepsy with ragged red fibers; or MERRF (mtDNA tRNA genes), and Dentatorubral-pallidoluysian atrophy or DRPLA (ATNI gene) are three severe forms of progressive myoclonus epilepsy. The corresponding gene defects have been identified and molecular diagnosis is now widely available. Recent advances in genetics and molecular biology offer promising venues toward the development of new therapies. For an expanded treatment of this topic see Jasper's Basic Mechanisms of the Epilepsies, Fourth Edition (Noebels JL, Avoli M, Rogawski MA, Olsen RW, Delgado-Escueta AV, eds) published by Oxford University Press (available on the National Library of Medicine Bookshelf [NCBI] at www.ncbi.nlm.nih.gov/ books).

Summary Laforin or malin deficiency causes Lafora disease, characterized by altered glycogen metabolism and teenage-onset neurodegeneration with intractable and invariably fatal epilepsy. Plant starches possess small amounts of metabolically essential monophosphate esters. Glycogen contains similar phosphate amounts, which are thought to originate from a glycogen synthase error side reaction and therefore lack any specific function. Glycogen is also believed to lack monophosphates at glucosyl carbon C6, an essential phosphorylation site in plant starch metabolism. We now show that glycogen phosphorylation is not due to a glycogen synthase side reaction, that C6 is a major glycogen phosphorylation site, and that C6 monophosphates predominate near centers of glycogen molecules and positively correlate with glycogen chain lengths. Laforin or malin deficiency causes C6 hyperphosphorylation, which results in malformed long-chained glycogen that accumulates in many tissues, causing neurodegeneration in brain. Our work advances the understanding of Lafora disease pathogenesis and suggests that glycogen phosphorylation has important metabolic function.

X-linked myopathy with excessive autophagy (XMEA) is a childhood-onset disease characterized by progressive vacuolation and atrophy of skeletal muscle. We show that XMEA is caused by hypomorphic alleles of the VMA21 gene, that VMA21 is the diverged human ortholog of the yeast Vma21p protein, and that like Vma21p it is an essential assembly chaperone of the V-ATPase, the principal mammalian proton pump complex. Decreased VMA21 raises lysosomal pH, which reduces lysosomal degradative ability and blocks autophagy. This reduces cellular free amino acids, which upregulates the mTOR pathway and mTOR-dependent macroautophagy, resulting in proliferation of large and ineffective autolysosomes that engulf sections of cytoplasm, merge together, and vacuolate the cell. Our results uncover macroautophagic overcompensation leading to cell vacuolation and tissue atrophy as a mechanism of disease.

In a mouse mutagenesis screen, we isolated a mutant, Myshkin ( Myk ), with autosomal dominant complex partial and secondarily generalized seizures, a greatly reduced threshold for hippocampal seizures in vitro, posttetanic hyperexcitability of the CA3-CA1 hippocampal pathway, and neuronal degeneration in the hippocampus. Positional cloning and functional analysis revealed that Myk /+ mice carry a mutation (I810N) which renders the normally expressed Na + ,K + -ATPase α3 isoform inactive. Total Na + ,K + -ATPase activity was reduced by 42% in Myk /+ brain. The epilepsy in Myk /+ mice and in vitro hyperexcitability could be prevented by delivery of additional copies of wild-type Na + ,K + -ATPase α3 by transgenesis, which also rescued Na + ,K + -ATPase activity. Our findings reveal the functional significance of the Na + ,K + -ATPase α3 isoform in the control of epileptiform activity and seizure behavior.

Lafora disease is characterized by pathognomonic inclusions, Lafora bodies (LB), in neurons and other cell types. In skin, LB have been reported in either eccrine sweat glands or in apocrine sweat glands. The disease is caused by mutations in either the EPM2A gene or in a second yet-unknown gene. Here the authors determine whether a genotype-phenotype correlation exists between the genetic form of the disease and the skin cell type affected by LB formation. Also is described an important source of false positivity in the use of axillary biopsies for disease diagnosis.

One quadrillion synapses are laid in the first two years of postnatal construction of the human brain, which are then pruned until age 10 to 500 trillion synapses composing the final network. Genetic epilepsies are the most common neurological diseases with onset during pruning, affecting 0.5% of 2–10-year-old children, and these epilepsies are often characterized by spontaneous remission. We previously described a remitting epilepsy in the Lagotto romagnolo canine breed. Here, we identify the gene defect and affected neurochemical pathway. We reconstructed a large Lagotto pedigree of around 34 affected animals. Using genome-wide association in 11 discordant sib-pairs from this pedigree, we mapped the disease locus to a 1.7 Mb region of homozygosity in chromosome 3 where we identified a protein-truncating mutation in the Lgi2 gene, a homologue of the human epilepsy gene LGI1. We show that LGI2, like LGI1, is neuronally secreted and acts on metalloproteinase-lacking members of the ADAM family of neuronal receptors, which function in synapse remodeling, and that LGI2 truncation, like LGI1 truncations, prevents secretion and ADAM interaction. The resulting epilepsy onsets at around seven weeks (equivalent to human two years), and remits by four months (human eight years), versus onset after age eight in the majority of human patients with LGI1 mutations. Finally, we show that Lgi2 is expressed highly in the immediate post-natal period until halfway through pruning, unlike Lgi1, which is expressed in the latter part of pruning and beyond. LGI2 acts at least in part through the same ADAM receptors as LGI1, but earlier, ensuring electrical stability (absence of epilepsy) during pruning years, preceding this same function performed by LGI1 in later years. LGI2 should be considered a candidate gene for common remitting childhood epilepsies, and LGI2-to-LGI1 transition for mechanisms of childhood epilepsy remission., Author Summary Major remodeling of the neuronal synaptic network occurs during childhood. The quadrillion synapses formed till the end of age two are trimmed to 500 trillion by age 10 through a selective process of strengthening of ideal connections, removal of redundant ones, and formation of new contacts. Very little is known about the basic mechanisms that direct this massive reorganization that leads to the adult brain. The most common epilepsies of humans occur in childhood and are characterized by remission prior to adulthood. Not much is known about their genetics and basic remission mechanisms. We describe here a canine equivalent disease and identify the defective gene, Lgi2. We show that the gene product is a secreted protein and interacts with neuronal ADAM receptors known to be involved in the regulation of synaptic remodeling in the developing brain. Our work sheds important light on the basic mechanisms of the most common neurological disease of children and discloses processes of epilepsy remission. The identification of the first focal epilepsy gene in dogs has also enabled the development of a genetic test to identify carriers for breeding purposes.

Lafora disease is a teenage onset progressive myoclonus epilepsy caused by mutations in the EPM2A gene. In this report, we describe new mutations within EPM2A, review the known mutations to date to identify the most common, and describe three simple tests for prenatal and carrier screening.