Two novel mutations in the <Emphasis Type="Italic">EPM2A </Emphasis>gene in a Korean patient with Lafora's progressive myoclonus epilepsy

 The progressive myoclonus epilepsy of the Lafora type (LD; MIM 254780) is a rare autosomal recessive disorder characterized by epilepsy, myoclonus, progressive neurological deterioration, and the presence of periodic acid-Schiff-positive polyglucosan inclusions (Lafora bodies). Mutations in the EPM2A gene have recently been found to cause LD and about 30 or more mutations have been reported thus far. LD is relatively common in countries of the Mediterranean Basin, the Middle East, India, and Pakistan. Although a few sporadic cases with the typical LD phenotype have also been reported in the Far East including Korea and Japan, a recent effort to find mutations in Japanese LD families was not successful. In the present study, we report two novel mutations in a Korean girl with LD; a 1-bp insertion mutation (c.223insC; G75fsX107) in exon 1 and a missense mutation (c.559A>G; T187A) in exon 3 of the EPM2A gene. To our knowledge, this is the first report of a genetically confirmed case of LD in Koreans and also in the Far East. Received: September 2, 2002 / Accepted: November 8, 2002 Acknowledgments This work was supported by the fund from the Center for Functional Analysis of Human Genome (FG-2–1-02), Korea. The third and sixth authors contributed equally to this work. Correspondence to:J.-W. Kim     

Mutational spectrum of the EPM2A gene in progressive myoclonus epilepsy of Lafora: high degree of allelic heterogeneity and prevalence of deletions

Progressive myoclonus epilepsy of the Lafora type (Lafora disease) is an autosomal recessive disease characterised by epilepsy, myoclonus, progressive neurological deterioration and the presence of glycogen-like intracellular inclusion bodies (Lafora bodies). We recently cloned the major gene for Lafora disease (EPM2A) and characterised the corresponding product, a putative protein tyrosine phosphatase (LAFPTPase). Here we report the complete coding sequence of the EPM2A gene and the analysis of this gene in 68 Lafora disease chromosomes. We describe 11 novel mutations: three missense (F84L, G240S and P301L), one nonsense (Y86stop), three < 40 bp microdeletions (K90fs, Ex1-32bpdel, Ex1-33bpdel), and two deletions affecting the entire exon 1 (Ex1-del1 and Ex1-del2). In addition, we have identified three patients with a null allele in non-exonic microsatellites EPM2A-3 or EPM2A-4, suggesting the presence of two distinct > 3 kb deletions affecting exon 2 (Ex2-del1 and Ex2-del2). Considering these mutations, a total of 25 mutations, 60% of them generating truncations, have been described thus far in the EPM2A gene. In spite of this remarkable allelic heterogeneity, the R241stop EPM2A mutation was found in approximately 40% of the Lafora disease patients. We also report the characterisation of five new microsatellite markers and one SNP in the EPM2A gene and describe the haplotypic associations of alleles at these sites in normal and EPM2A chromosomes. This analysis suggests that both founder effect and recurrence have contributed to the relatively high prevalence of R241stop mutation in Spain. The data reported here represent the first systematic analysis of the mutational events in the EPM2A gene in Lafora disease patients and provide insight into the origin and evolution of the different EPM2A alleles.     

A novel protein tyrosine phosphatase gene is mutated in progressive myoclonus epilepsy of the Lafora type (EPM2)

Progressive myoclonus epilepsy of the Lafora type or Lafora disease (EPM2; McKusick no. 254780) is an autosomal recessive disorder characterized by epilepsy, myoclonus, progressive neurological deterioration and glycogen-like intracellular inclusion bodies (Lafora bodies). A gene for EPM2 previously has been mapped to chromosome 6q23- q25 using linkage analysis and homozygosity mapping. Here we report the positional cloning of the 6q EPM2 gene. A microdeletion within the EPM2 critical region, present inhomozygosis in an affected individual, was found to disrupt a novel gene encoding a putative protein tyrosine phosphatase (PTPase). The gene, denoted EPM2, presents alternative splicing in the 5' and 3' end regions. Mutational analysis revealed that EPM2 patients are homozygous for loss-of-function mutations in EPM2. These findings suggest that Lafora disease results from the mutational inactivation of a PTPase activity that may be important in the control of glycogen metabolism.      

Mutations in the NHLRC1 gene are the common cause for Lafora disease in the Japanese population

Lafora disease (LD) is a rare autosomal recessive genetic disorder characterized by epilepsy, myoclonus, and progressive neurological deterioration. LD is caused by mutations in the EMP2A gene encoding a protein phosphatase. A second gene for LD, termed NHLRC1 and encoding a putative E3 ubiquitin ligase, was recently identified on chromosome 6p22. The LD is relatively common in southern Europe, the Middle East, and Southeast Asia. A few sporadic cases with typical LD phenotype have been reported from Japan; however, our earlier study failed to find EPM2A mutations in four Japanese families with LD. We recruited four new families from Japan and searched for mutations in EPM2A . All eight families were also screened for NHLRC1 mutations. We found five independent families having novel mutations in NHLRC1. Identified mutations include five missense mutations (p.I153M, p.C160R, p.W219R, p.D245N, and p.R253K) and a deletion mutation (c.897insA; p.S299fs13). We also found a family with a ten base pair deletion (c.822-832del10) in the coding region of EPM2A. In two families, no EPM2A or NHLRC1 mutation was found. Our study, in addition to documenting the genetic and molecular heterogeneity observed for LD, suggests that mutations in the NHLRC1 gene may be a common cause of LD in the Japanese population.     

Lafora disease in the Indian population: EPM2A and NHLRC1 gene mutations and their impact on subcellular localization of laforin and malin

Lafora disease (LD) is a fatal form of teenage-onset autosomal recessive progressive myoclonus epilepsy. LD is more common among geographic isolates and in populations with a higher rate of consanguinity. Mutations in two genes, EPM2A encoding laforin phosphatase, and NHLRC1 encoding malin ubiquitin ligase, have been shown to cause the LD. We describe here a systematic analysis of the EPM2A and the NHLRC1 gene sequences in 20 LD families from the Indian population. We identified 12 distinct mutations in 15 LD families. The identified novel mutations include 4 missense mutations (K140N, L310W, N148Y, and E210 K) and a deletion of exon 3 for EPM2A, and 4 missense mutations (S22R, L279P, L279P, and L126P) and a single base-pair insertional mutation (612insT) for NHLRC1. The EPM2A gene is known to encode two laforin isoforms having distinct carboxyl termini; a major isoform localized in the cytoplasm, and a minor isoform that targeted the nucleus. We show here that the effect of the EPM2A gene mutation L310W was limited to the cytoplasmic isoform of laforin, and altered its subcellular localization. We have also analyzed the impact of NHLRC1 mutations on the subcellular localization of malin. Of the 6 distinct mutants tested, three targeted the nucleus, one formed perinuclear aggregates, and two did not show any significant difference in the subcellular localization as compared to the wild-type malin. Our results suggest that the altered subcellular localization of mutant proteins of the EPM2A and NHLRC1 genes could be one of the molecular bases of the LD phenotype.     

Lafora progressive myoclonus epilepsy: NHLRC1 mutations affect glycogen metabolism

Lafora disease is a fatal autosomal recessive form of progressive myoclonus epilepsy. Patients manifest myoclonus and tonic-clonic seizures, visual hallucinations, intellectual, and progressive neurologic deterioration beginning in adolescence. The two genes known to be involved in Lafora disease are EPM2A and NHLRC1 (EPM2B). The EPM2A gene encodes laforin, a dual-specificity protein phosphatase, and the NHLRC1 gene encodes malin, an E3-ubiquitin ligase. The two proteins interact with each other and, as a complex, are thought to regulate glycogen synthesis. Here, we report three Lafora families with two novel pathogenic mutations (C46Y and L261P) and two recurrent mutations (P69A and D146N) in NHLRC1. Investigation of their functional consequences in cultured mammalian cells revealed that malin(C46Y), malin(P69A), malin(D146N), and malin(L261P) mutants failed to downregulate the level of R5/PTG, a regulatory subunit of protein phosphatase 1 involved in glycogen synthesis. Abnormal accumulation of intracellular glycogen was observed with all malin mutants, reminiscent of the polyglucosan inclusions (Lafora bodies) present in patients with Lafora disease.     

Laforin, the dual-phosphatase responsible for Lafora disease, interacts with R5 (PTG), a regulatory subunit of protein phosphatase-1 that enhances glycogen accumulation

Progressive myoclonus epilepsy of Lafora type (LD, MIM 254780) is a fatal autosomal recessive disorder characterized by the presence of progressive neurological deterioration, myoclonus, epilepsy and polyglucosan intracellular inclusion bodies, called Lafora bodies. Lafora bodies resemble glycogen with reduced branching, suggesting an alteration in glycogen metabolism. Linkage analysis and homozygosity mapping localized EPM2A, a major gene for LD, to chromosome 6q24. EPM2A encodes a protein of 331 amino acids (named laforin) with two domains, a dual-specificity phosphatase domain and a carbohydrate binding domain. Here we show that, in addition, laforin interacts with itself and with the glycogen targeting regulatory subunit R5 of protein phosphatase 1 (PP1). R5 is the human homolog of the murine Protein Targeting to Glycogen, a protein that also acts as a molecular scaffold assembling PP1 with its substrate, glycogen synthase, at the intracellular glycogen particles. The laforin-R5 interaction was confirmed by pull-down and co-localization experiments. Full-length laforin is required for the interaction. However, a minimal central region of R5 (amino acids 116-238), including the binding sites for glycogen and for glycogen synthase, is sufficient to interact with laforin. Point-mutagenesis of the glycogen synthase-binding site completely blocked the interaction with laforin. The majority of the EPM2A missense mutations found in LD patients result in lack of phosphatase activity, absence of binding to glycogen and lack of interaction with R5. Interestingly, we have found that the LD-associated EPM2A missense mutation G240S has no effect on the phosphatase or glycogen binding activities of laforin but disrupts the interaction with R5, suggesting that binding to R5 is critical for the laforin function. These results place laforin in the context of a multiprotein complex associated with intracellular glycogen particles, reinforcing the concept that laforin is involved in the regulation of glycogen metabolism.     

Genotype-phenotype correlations for EPM2A mutations in Lafora's progressive myoclonus epilepsy: exon 1 mutations associate with an early-onset cognitive deficit subphenotype

Mutations in the EPM2A gene encoding a dual-specificity phosphatase (laforin) cause an autosomal recessive fatal disorder called Lafora's disease (LD) classically described as an adolescent-onset stimulus-sensitive myoclonus, epilepsy and neurologic deterioration. Here we related mutations in EPM2A with phenotypes of 22 patients (14 families) and identified two subsyndromes: (i) classical LD with adolescent-onset stimulus-sensitive grand mal, absence and myoclonic seizures followed by dementia and neurologic deterioration, and associated mainly with mutations in exon 4 (P = 0.0007); (ii) atypical LD with childhood-onset dyslexia and learning disorder followed by epilepsy and neurologic deterioration, and associated mainly with mutations in exon 1 (P = 0.0015). To understand the two subsyndromes better, we investigated the effect of five missense mutations in the carbohydrate-binding domain (CBD-4; coded by exon 1) and three missense mutations in the dual phosphatase domain (DSPD; coded by exons 3 and 4) on laforin's intracellular localization in HeLa cells. Expression of three mutant proteins (T194I, G279S and Y294N) in DSPD formed ubiquitin-positive cytoplasmic aggregates, suggesting that they were folding mutants set for degradation. In contrast, none of the three CBD-4 mutants showed cytoplasmic clumping. However, CBD-4 mutants W32G and R108C targeted both cytoplasm and nucleus, suggesting that laforin had diminished its usual affinity for polysomes. Our data, thus, represent the first report of a novel childhood syndrome for LD. Our results also provide clues for distinct roles for the CBD-4 and DSP domains of laforin in the etiology of two subsyndromes of LD.     

Lafora progressive myoclonus epilepsy: A meta‐analysis of reported mutations in the first decade following the discovery of the EPM2A and NHLRC1 genes

Lafora disease (LD) is an autosomal recessive and fatal form of progressive myoclonus epilepsy. LD patients manifest myoclonus and tonic–clonic seizures, visual hallucinations, and progressive neurologic deterioration beginning at 12 to 15 years of age. The two genes known to be associated with LD are EPM2A and NHLRC1. Mutations in at least one other as yet unknown gene also cause LD. The EMP2A encodes a protein phosphatase and NHLRC1 encodes an ubiquitin ligase. These two proteins interact with each other and, as a complex, are thought to regulate critical neuronal functions. Nearly 100 distinct mutations have been discovered in the two genes in over 200 independent LD families. Nearly half of them are missense mutations, and the deletion mutations account for one‐quarter. Several reports have provided functional data for the mutant proteins and a few also provide genotype–phenotype correlations. In this review we provide an update on the spectrum of EPM2A and NHLRC1 mutations, and discuss their distribution in the patient population, genotype–phenotype correlations, and on the possible effect of disease mutations on the cellular functions of LD proteins. Hum Mutat 0, 1–9, 2009. © 2009 Wiley‐Liss, Inc.     

Regulation of glycogen synthesis by the laforin-malin complex is modulated by the AMP-activated protein kinase pathway

Lafora progressive myoclonus epilepsy (LD) is a fatal autosomal recessive neurodegenerative disorder characterized by the presence of glycogen-like intracellular inclusions called Lafora bodies. LD is caused by mutations in two genes, EPM2A and EPM2B, encoding respectively laforin, a dual-specificity protein phosphatase, and malin, an E3 ubiquitin ligase. Previously, we and others have suggested that the interactions between laforin and PTG (a regulatory subunit of type 1 protein phosphatase) and between laforin and malin are critical in the pathogenesis of LD. Here, we show that the laforin-malin complex downregulates PTG-induced glycogen synthesis in FTO2B hepatoma cells through a mechanism involving ubiquitination and degradation of PTG. Furthermore, we demonstrate that the interaction between laforin and malin is a regulated process that is modulated by the AMP-activated protein kinase (AMPK). These findings provide further insights into the critical role of the laforin-malin complex in the control of glycogen metabolism and unravel a novel link between the energy sensor AMPK and glycogen metabolism. These data advance our understanding of the functional role of laforin and malin, which hopefully will facilitate the development of appropriate LD therapies.     

Phenotype variations in Lafora progressive myoclonus epilepsy: possible involvement of genetic modifiers?

Lafora progressive myoclonus epilepsy, also known as Lafora disease (LD), is the most severe and fatal form of progressive myoclonus epilepsy with its typical onset during the late childhood or early adolescence. LD is characterized by recurrent epileptic seizures and progressive decline in intellectual function. LD can be caused by defects in any of the two known genes and the clinical features of these two genetic groups are almost identical. The past one decade has witnessed considerable success in identifying the LD genes, their mutations, the cellular functions of gene products and on molecular basis of LD. Here, we briefly review the current literature on the phenotype variations, on possible presence of genetic modifiers, and candidate modifiers as targets for therapeutic interventions in LD.     

Clinical and genetic data on Lafora disease patients of Serbian/Montenegrin origin

Lafora disease (LD) is an autosomal recessive, progressive disorder characterized by myoclonus and seizures, inexorable neurologic deterioration, cognitive decline and poor prognosis. LD is caused by mutations either in the EPM2A or in NHLRC1 genes. Here we report clinical and genetic findings on 14 LD patients from 10 families of Serbian/Montenegrin origin. Molecular diagnostics was performed by sequencing the coding regions of the EPM2A and NHLRC1 genes. In addition, haplotype analysis of the chromosomes carrying the two most frequent mutations (c.1048‐1049delGA and deletion of the whole NHLRC1 gene) using eight different markers flanking the NHLRC1 gene was conducted. We identified one new mutation (c.1028T>C) along with the 3 previously reported mutations (c.1048‐1049delGA, c.990delG, deletion of the whole NHLRC1 gene), all of which were located on the NHLRC1 gene. The two predominant mutations (c.1048‐1049delGA and complete NHLRC1 gene deletion) appear to be founder mutations. In addition to documenting the genetic heterogeneity observed for LD, our study suggests that mutations in the NHLRC1 gene may be a common cause of LD in the Serbian/Montenegrin population, primarily because of a founder effect.     

Laforin, defective in the progressive myoclonus epilepsy of Lafora type, is a dual-specificity phosphatase associated with polyribosomes

The progressive myoclonus epilepsy of Lafora type is an autosomal recessive disorder caused by mutations in the EPM2A gene. EPM2A is predicted to encode a putative tyrosine phosphatase protein, named laforin, whose full sequence has not yet been reported. In order to understand the function of the EPM2A gene, we isolated a full-length cDNA, raised an antibody and characterized its protein product. The full-length clone predicts a 38 kDa laforin that was very close to the size detected in transfected cells. Recombinant laforin was able to hydrolyze phosphotyrosine as well as phosphoserine/threonine substrates, demonstrating that laforin is an active dual-specificity phosphatase. Biochemical, immunofluorescence and electron microscopic studies on the full-length laforin expressed in HeLa cells revealed that laforin is a cytoplasmic protein associated with polyribosomes, possibly through a conformation-dependent protein-protein interaction. We analyzed the intracellular targeting of two laforin mutants with missense mutations. Expression of both mutants resulted in ubiquitin-positive perinuclear aggregates suggesting that they were misfolded proteins targeted for degradation. Our results suggest that laforin is involved in translational regulation and that protein misfolding may be one of the molecular bases of the Lafora disease phenotype caused by missense mutations in the EPM2A gene.     

Recent advances in the molecular basis of Lafora’s progressive myoclonus epilepsy

Laforas disease (LD) is an autosomal recessive and fatal form of progressive myoclonus epilepsy with onset in late childhood or adolescence. LD is characterised by the presence of intracellular polyglucosan inclusions, called Lafora bodies, in tissues including the brain, liver and skin. Patients have progressive neurologic deterioration, leading to death within 10 years of onset. No preventive or curative treatment is available for LD. At least three genes underlie LD, of which two have been isolated and mutations characterised: EPM2A and NHLRC1. The EPM2A gene product laforin is a protein phosphatase while the NHLRC1 gene product malin is an E3 ubiquitin ligase that ubiquitinates and promotes the degradation of laforin. Analyses of the structure and function of these gene products suggest defects in post-translational modification of proteins as the common mechanism that leads to the formation of Lafora inclusion bodies, neurodegeneration and the epileptic phenotype of LD. In this review, we summarise the available information on the genetic basis of LD, and correlate these advances with the rapidly expanding information about the mechanisms of LD gained from studies on both cell biological and animal models. Finally, we also discuss a possible mechanism to explain the locus heterogeneity observed in LD.     

The Lafora disease gene product laforin interacts with HIRIP5, a phylogenetically conserved protein containing a NifU-like domain

Lafora disease is an autosomal recessive type of progressive myoclonus epilepsy caused by mutations in the EPM2A gene. The EPM2A gene-encoded protein laforin is a dual-specificity phosphatase that associates with polyribosomes. Because the cellular functions of laforin are largely unknown, we used the yeast-two hybrid system to screen for protein(s) that interact with laforin. We found that laforin interacts with a phylogenetically conserved protein HIRIP5 that harbors a NifU-like domain. Both in vitro and in vivo assay have shown that the interaction is specific and that laforin probably uses its N-terminal CBD-4 domain to interact with the C-terminal NifU-like domain of the HIRIP5 protein. HIRIP5 encodes a cytosolic protein and is expressed ubiquitously, perhaps reflecting a house-keeping function. The presence of a NifU-like domain in the HIRIP5 protein raises an interesting possibility that it may be involved in iron homeostasis. Although the significance of the interaction between HIRIP5 and laforin proteins is not yet fully known, because laforin dephosphorylated HIRIP5 in vitro, HIRIP5 promises to be an interesting laforin-binding partner and would contribute to the understanding of the molecular pathology of Lafora disease.     

Targeted disruption of the Epm2a gene causes formation of Lafora inclusion bodies,neurodegeneration, ataxia,myoclonus epilepsy and impaired behavioral response in mice

Mutations in the EPM2A gene encoding a dual-specificity phosphatase (laforin) cause Lafora disease (LD), a progressive and invariably fatal epilepsy with periodic acid-Schiff-positive (PAS+) cytoplasmic inclusions (Lafora bodies) in the central nervous system. To study the pathology of LD and the functions of laforin, we disrupted the Epm2a gene in mice. At two months of age, homozygous null mutants developed widespread degeneration of neurons, most of which occurred in the absence of Lafora bodies. Dying neurons characteristically exhibit swelling in the endoplasmic reticulum, Golgi networks and mitochondria in the absence of apoptotic bodies or fragmentation of DNA. As Lafora bodies become more prominent at 4-12 months, organelles and nuclei are disrupted. The Lafora bodies, present both in neuronal and non-neural tissues, are positive for ubiquitin and advanced glycation end-products only in neurons, suggesting different pathological consequence for Lafora inclusions in neuronal tissues. Neuronal degeneration and Lafora inclusion bodies predate the onset of impaired behavioral responses, ataxia, spontaneous myoclonic seizures and EEG epileptiform activity. Our results suggest that LD is a primary neurodegenerative disorder that may utilize a non-apoptotic mechanism of cell death.     

Lafora disease: a case report, pathologic and genetic study

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.     

Dysfunctions in endosomal-lysosomal and autophagy pathways underlie neuropathology in a mouse model for Lafora disease

Lafora progressive myoclonus epilepsy (also known as Lafora disease, LD) is an inherited and fatal form of a neurodegenerative disorder characterized by the presence of carbohydrate-rich inclusions called Lafora bodies. LD can be caused by defects in the laforin phosphatase or the malin ubiquitin ligase and the clinical symptoms resulting from these two defects are almost similar. In order to understand the molecular basis of LD pathogenesis and the role of Lafora bodies in neuropathology, we have studied the laforin-deficient mice as a model and show here that Lafora bodies recruit proteasomal subunit, endoplasmic reticulum chaperone GRP78/Bip, autophagic protein p62 and endosomal regulators Rab5 and Rab7. The laforin-deficient brain also reveals the proliferation of enlarged lysosomes, lipofuscin granules, amyloid-β peptides and increased levels of insoluble form of ubiquitinated protein, indicating a significant impairment in the cellular degradative pathway. Further, abnormal dendrites and increased gliosis, especially at the vicinity of Lafora bodies, were noted in the LD brain. Taken together, our study suggests that the neuropathology in LD is not limited to Lafora bodies, that some of the neuropathological changes in LD are likely to be secondary effects caused by Lafora bodies, and that impairment in the autophagy-endosomal-lysosomal pathways might underlie some of the symptoms in LD.