EPM2A Mutations Research Articles

Reversible Glucan Phosphorylation Requires Unique Carbohydrate Binding Platforms

Research into starch metabolism and the fatal neurodegenerative epilepsy called Lafora disease are surprisingly linked by a family of enzymes that we recently discovered called glucan phosphatases. Plants release the energy in transitory starch via a recently identified three‐step process: starch phosphorylation, degradation, and dephosphorylation. Dikinases phosphorylate the outer starch glucose units to make them water‐soluble and enzyme accessible so that amylases can release maltose and glucose. Following amylase activity, the phosphate must be removed by glucan phosphatases. In the absence of glucan phosphates, plants cannot access the energy stored in starch and the starch granules grow in size while plant growth is stunted.We recently determined the structure of the Arabidopsis glucan phosphatases Starch Excess 4 (SEX4) and Like Sex Four2 (LSF2) with phospho‐glucan product bound at 1.62Å and 2.3Å, respectively. The glucose moiety closest to the SEX4 active site is positioned with the oxygen of the C6 carbon directed into the catalytic cleft and we find that SEX4 preferentially dephosphorylates C6 hydroxyls. Alternatively, the glucose moiety closest to the LSF2 active site is positioned with the C3 carbon towards the catalytic cleft and we find that LSF2 exclusively dephosphorylates C3 hydroxyls. Using structural and biochemical insights, we completely reversed SEX4 specificity, providing a method for engineering glucan phosphatase activity.The human EPM2A gene encodes laforin and recessive mutations in EPM2A result in Lafora disease (LD). In the absence of laforin activity, glycogen transforms into a hyper‐phosphorylated, water‐insoluble, starch‐like Lafora body (LB) that drives neuronal apoptosis, neurodegeneration, and eventual death of LD patients. The physiological function of laforin is to dephosphorylate glycogen, yet the mechanism of glycogen dephosphorylation by laforin was unknown. Additionally, LD missense mutations are dispersed throughout laforin, bringing to question the structural mechanism(s) of disease. We determined the crystal structure of human laforin at 2.4 Å bound to oligosaccharides with a phospho‐glucan product at the active site. The structure reveals an integrated tertiary structure of the carbohydrate binding module and dual specificity phosphatase domains as well as an antiparallel dimer mediated by the phosphatase domain that results in a tetramodular architecture, positioning the two active sites ~31 Å from each other. We utilized the crystal structure and three solution‐based, biophysical techniques along with biochemical analyses of LD patient mutations and structured guided mutations to probe this unique tertiary and quaternary structure. We define a cooperative mechanism of action for laforin as well as establish the effect of LD disease mutations, thereby providing atomic level insights that connect basic glycogen metabolism to human neurodegenerative disease.These structures are the first of glucan phosphatases, and they provide new insights into the molecular basis of this medically‐, agriculturally‐, and industry‐relevant enzyme family as well as their unique mechanisms of catalysis, substrate specificity, and interaction with glucans.Support or Funding InformationNIH Grants R01NS070899 and P20GM103486; NSF grant MCB‐1252345; KSEF Grants KSEF‐2268‐RDE‐014 and KSEF‐2971‐RDE‐ 017; Mizutani Foundation for Glycoscience Award

PP01.12 – 2602: Differential diagnosis of progressive myoclonic epilepsia: Five patients with Lafora disease

Objective Lafora disease (LD) is a rare, fatal, autosomal recessive hereditary disease characterized by epilepsy, myoclonus and progressive neurological deterioration. Diagnosis is made by polyglucosan inclusion bodies (Lafora bodies) shown in skin biopsy. Responsible mutations involves either the EPM2A or NHLRC1 (EPM2B) gene. Methods We report genetic mutations and clinical courses of Lafora disease in 5 adolescents with homozygote NHLRC1 mutation and homozygous EPM2A mutation. Results A total 5 patients (3 female, 2 male) were presented with progressive myoclonic epilepsy. In all cases there were no problems at birth, childhood, and their psychomotor development were normal and attended regular school. In general seizures began around at 13–14 years old. Over the time they demostrated ataxia, and significant deterioration occurred in mental functions, myoclonus of the extremities started, and 3 of all patients were unable to walk unassisted and one of them died at 15 years old. Axillary skin biopsies were performed to all and Lafora bodies in cytoplasm were demostrated. All patients parents are second-degree relatives. Routine blood test, cerebrospinal fluid analaysis were normal. In three patients cranial magnetic resonance imaging (MRI) were normal, but 2 patients had mild serebral and serebellar atrophy. There were multifocal, from time-to-time generalized spike-wave activity were observed in their electroencephalography (EEG). Genetic analaysis for Lafora was studied and 2 patients (sublings) had same mutation) homozygous (c.336 C>A) p.Y112X mutation in EPM2A gene, homozygous (c.199 G>T) p. E67X mutation (2 patient had same mutation) in NHLRC1 gene, homozygous L 180V (CTG>GTC) mutation in EPM2A gene were detected. Conclusion LD is characterized by progressive myoclonic epilepsy and neurodegenerative symptoms. Therefore early recognation is diffucult. If patient show progressive myoclonic seizures with progressive mental deterotation, as well as ataxi we should consider LD

P77 - Increased oxidative stress and impaired antioxidant response in Lafora disease

Lafora Disease (LD, OMIM 254780, ORPHA501) is a fatal neurodegenerative disorder characterized by the presence of glycogen-like intracellular inclusions called Lafora bodies and caused, in the vast majority of cases, by mutations in either EPM2A or EPM2B genes, encoding respectively laforin and malin. In the last years, several reports have revealed molecular details of these two proteins and have identified several processes affected in LD, but the pathophysiology of the disease still remains largely unknown. Since autophagy impairment has been reported as a characteristic treat in both Lafora disease cell and animal models, and as there is a link between autophagy and mitochondrial performance, we sought to determine if mitochondrial function could be altered in those models. Using fibroblasts from LD patients, deficient in laforin or malin, we found mitochondrial alterations, oxidative stress and a deficiency in antioxidant enzymes involved in the detoxification of reactive oxygen species (ROS). Similar results were obtained in brain tissue samples from transgenic mice deficient in either the EPM2A or EPM2B genes. Furthermore, in a proteomic analysis of brain tissue obtained from Epm2b-/- mice, we observed an increase in a modified form of peroxirredoxin-6, an antioxidant enzyme involved in other neurological pathologies, thus corroborating an alteration of the redox condition. These data support that oxidative stress produced by an increase in ROS production and an impairment of the antioxidant enzyme response to this stress play an important role in development of LD.

Mild Lafora disease: clinical, neurophysiologic, and genetic findings.

We report clinical, neurophysiologic, and genetic features of an Italian series of patients with Lafora disease (LD) to identify distinguishing features of those with a slowly progressive course. Twenty-three patients with LD (17 female; 6 male) were recruited. Mean age (± SD) at the disease onset was 14.5±3.9years and mean follow-up duration was 13.2±8.0years. NHLRC1 mutations were detected in 18 patients; EPM2A mutations were identified in 5. Patients who maintained >10years gait autonomy were labeled as "mild" and were compared with the remaining LD patients with a typical course. Six of 23 patients were mild and presented significantly delay in the age at onset, lower neurologic disability score at 4years after the onset, less severe seizure phenotype, lower probability of showing both photoparoxysmal response on electroencephalography (EEG) and giant somatosensory evoked potentials, as compared to patients with typical LD. However, in both mild and typical LD patients, EEG showed disorganization of background activity and frequent epileptiform abnormalities. Mild LD patients had NHLRC1 mutations and five of six carried homozygous or compound heterozygous D146N mutation. This mutation was found in none of the patients with typical LD. The occurrence of specific NHLRC1 mutations in patients with mild LD should be taken into account in clinical practice for appropriate management and counseling.

Late onset Lafora disease and novel EPM2A mutations: Breaking paradigms

Lafora disease (LD) is an autosomal recessive progressive myoclonus epilepsy with classic adolescent onset of stimuli sensitive seizures. Patients typically deteriorate rapidly with dementia, ataxia, vegetative failure and death by 25 years of age. LD is caused by homozygous mutations in EPM2A or EPM2B genes. We found four novel mutations in EPM2A - three in exon 4 (Q247X, H265R G279C) and one in exon 1 (Y86D) - and a previously described mutation in exon 4 (R241X). These five EPM2A mutations were found in four index cases and affected relatives. Patient 1 with classic LD was doubly heterozygous for H265R and R241X in exon 4; while Patient 2, who also had classic LD, was homozygous for Q247X in exon 4. Patient 3 with classic LD was homozygous for Y86D in exon 1, but the same mutation in his affected brother manifested an atypical earlier childhood onset. For the first time, we describe a later onset and slower progression of EPM2A-deficient LD seen in Patient 4 and her three sisters who were doubly heterozygous for R241X and G279C in exon 4. In these sisters, seizures started later at 21 to 28 years of age and progressed slowly with patients living beyond 30 years of age. Our observations suggest that variations in phenotypes of EPM2A-deficient LD, like an earlier childhood or adolescent or later adult onset with a rapid or slower course, depend on a second modifying factor separate from pathogenicity or exon location of EPM2A mutations. A modifying gene amongst the patient's genetic background or environmental factors may condition age of onset and rapid or slow progression of LD.

Increased Oxidative Stress and Impaired Antioxidant Response in Lafora Disease

Lafora disease (LD, OMIM 254780, ORPHA501) is a fatal neurodegenerative disorder characterized by the presence of glycogen-like intracellular inclusions called Lafora bodies and caused, in the vast majority of cases, by mutations in either EPM2A or EPM2B genes, encoding respectively laforin and malin. In the last years, several reports have revealed molecular details of these two proteins and have identified several processes affected in LD, but the pathophysiology of the disease still remains largely unknown. Since autophagy impairment has been reported as a characteristic treat in both Lafora disease cell and animal models, and as there is a link between autophagy and mitochondrial performance, we sought to determine if mitochondrial function could be altered in those models. Using fibroblasts from LD patients, deficient in laforin or malin, we found mitochondrial alterations, oxidative stress and a deficiency in antioxidant enzymes involved in the detoxification of reactive oxygen species (ROS). Similar results were obtained in brain tissue samples from transgenic mice deficient in either the EPM2A or EPM2B genes. Furthermore, in a proteomic analysis of brain tissue obtained from Epm2b-/- mice, we observed an increase in a modified form of peroxiredoxin-6, an antioxidant enzyme involved in other neurological pathologies, thus corroborating an alteration of the redox condition. These data support that oxidative stress produced by an increase in ROS production and an impairment of the antioxidant enzyme response to this stress play an important role in development of LD.

Identification of proteins potentially involved in the formation of Lafora bodies, a hallmark of Lafora disease

Background Lafora Disease (LD) is a fatal teenage-onset progressive myoclonus epilepsy. It is characterized by the formation of Lafora bodies (LBs), deposits of abnormally branched, insoluble, hyperphosphorylated glycogen-like polymers that are generally believed to trigger the development of the clinical symptoms of LD. 58% and 35% of the LD cases are caused by mutations in EPM2A (laforin) and EPM2B (malin), respectively. However, little is known about their function in LB formation. Two different mechanisms have been proposed to explain the accumulation of insoluble LBs: first, excessive glycogen phosphorylation and, second, an imbalance between glycogen synthesizing enzymes. The present study aims at the identification of proteins involved in the molecular mechanisms leading to LB formation and appearance of LD and the phosphorylation of glycogen.

Lafora disease: Severe phenotype associated with homozygous deletion of the NHLRC1 gene

Lafora disease (LD) is a severe, autosomal recessive, latechildhood- to teenage-onset, progressive myoclonic epilepsy. It is due to either EPM2A or NHLRC1 mutations. We describe a patient with homozygous deletion encompassing the entire NHLRC1 gene, not previously reported, and with clinical course more progressive than in the most patients with NHLRC1 mutations. The diagnosis of LD in our patient was based on the typical clinic, neurophysiological presentation, as well as skin biopsy followed by molecular genetics findings. She developed normally until the age of 15, when she had her first occipital and generalized seizures. Four years after the first seizure the patient became bedridden, demented and presented with severe clinical condition. She died of pneumonia at age 20. This report is the first case of homozygosity for NHLRC1 deletion and thus adds to mutational heterogeneity of LD. Besides, it widens the spectrum of LD patients with severe phenotype and NHLRC1 mutations.

Progressive myoclonic encephalopathies: From phenomenology to genes and proteins

The progressive myoclonic epilepsies (PMEs) are a group of symptomatic generalized epilepsies caused by rare disorders, most of which have a genetic component, a debilitating course, and a poor outcome. Challenges with PME arise from difficulty with diagnosis, especially in the early stages of the illness, and further problems of management and drug treatment. PMEs are characterized by myoclonic seizures, tonic–clonic seizures, and progressive neurological deterioration, typically with cerebellar signs and dementia; other signs however can be present especially during the progression of disease. Recent advances in molecular genetics have helped to achieve better understanding of the different disorders that cause PME. There are different forms of PME that have been more accurately defined with recent advances in genetic studies. In particular, especially in adult age, the most recognized forms included Unverricht–Lundborg disease (related to CSTB gene mutation formerly indicated as EPM1A), Lafora disease (related to EPM2A and EPM2B mutation), myoclonic epilepsy with ragged red fibers (a mitochondrial disease often related to MTTK mutation), neuronal ceroidolipofuscinoses (CLN2, CLN3, CLN5, CLN6), sialidoses (that include different forms secondary to neuraminidase deficiency) and dentatorubral-pallidoluysian atrophy (related to an abnormal CAG repeat). The paper is aimed to review the PMEs with emphasis on updated genetics, diagnosis, and therapeutic options.

Laforin is required for the functional activation of malin in endoplasmic reticulum stress resistance in neuronal cells

Mutations in either EPM2A, the gene encoding a dual-specificity phosphatase named laforin, or NHLRC1, the gene encoding an E3 ubiquitin ligase named malin, cause Lafora disease in humans. Lafora disease is a fatal neurological disorder characterized by progressive myoclonus epilepsy, severe neurological deterioration and accumulation of poorly branched glycogen inclusions, called Lafora bodies or polyglucosan bodies, within the cell cytoplasm. The molecular mechanism underlying the neuropathogenesis of Lafora disease remains unknown. Here, we present data demonstrating that in the cells expressing low levels of laforin protein, overexpressed malin and its Lafora disease-causing missense mutants are stably polyubiquitinated. Malin and malin mutants form ubiquitin-positive aggregates in or around the nuclei of the cells in which they are expressed. Neither wild-type malin nor its mutants elicit endoplasmic reticulum stress, although the mutants exaggerate the response to endoplasmic reticulum stress. Overexpressed laforin impairs the polyubiquitination of malin while it recruits malin to polyglucosan bodies. The recruitment and activities of laforin and malin are both required for the polyglucosan body disruption. Consistently, targeted deletion of laforin in brain cells from Epm2a knockout mice increases polyubiquitinated proteins. Knockdown of Epm2a or Nhlrc1 in neuronal Neuro2a cells shows that they cooperate to allow cells to resist ER stress and apoptosis. These results reveal that a functional laforin-malin complex plays a critical role in disrupting Lafora bodies and relieving ER stress, implying that a causative pathogenic mechanism underlies their deficiency in Lafora disease.

Four novel and two recurrent NHLRC1 (EPM2B) and EPM2A gene mutations leading to Lafora disease in six Turkish families

Lafora disease (LD) is a type of autosomal recessive, progressive myoclonus epilepsy resulting mostly from mutations in the EPM2A and NHLRC1 genes. Mutational analysis in both genes was initiated with the aim of establishing LD DNA diagnosis in Turkey. Four novel NHLRC1 (p.G131X, p.P69S and p.D82H) and EPM2A (p.V7A) and two recurrent NHLRC1 (p.D146N) and EPM2A (p.R241X) mutations were identified in six families. The delineation of causative mutations in patients provided early disease diagnosis for other family members and contributed to the knowledge of LD pathogenesis.

Neurodegeneration and functional impairments associated with glycogen synthase accumulation in a mouse model of Lafora disease

Lafora disease (LD) is caused by mutations in either the laforin or malin gene. The hallmark of the disease is the accumulation of polyglucosan inclusions called Lafora Bodies (LBs). Malin knockout (KO) mice present polyglucosan accumulations in several brain areas, as do patients of LD. These structures are abundant in the cerebellum and hippocampus. Here, we report a large increase in glycogen synthase (GS) in these mice, in which the enzyme accumulates in LBs. Our study focused on the hippocampus where, under physiological conditions, astrocytes and parvalbumin-positive (PV+) interneurons expressed GS and malin. Although LBs have been described only in neurons, we found this polyglucosan accumulation in the astrocytes of the KO mice. They also had LBs in the soma and some processes of PV+ interneurons. This phenomenon was accompanied by the progressive loss of these neuronal cells and, importantly, neurophysiological alterations potentially related to impairment of hippocampal function. Our results emphasize the relevance of the laforin–malin complex in the control of glycogen metabolism and highlight altered glycogen accumulation as a key contributor to neurodegeneration in LD.

A PTG Variant Contributes to a Milder Phenotype in Lafora Disease

Lafora disease is an autosomal recessive form of progressive myoclonus epilepsy with no effective therapy. Although the outcome is always unfavorable, onset of symptoms and progression of the disease may vary. We aimed to identify modifier genes that may contribute to the clinical course of Lafora disease patients with EPM2A or EPM2B mutations. We established a list of 43 genes coding for proteins related to laforin/malin function and/or glycogen metabolism and tested common polymorphisms for possible associations with phenotypic differences using a collection of Lafora disease families. Genotype and haplotype analysis showed that PPP1R3C may be associated with a slow progression of the disease. The PPP1R3C gene encodes protein targeting to glycogen (PTG). Glycogen targeting subunits play a major role in recruiting type 1 protein phosphatase (PP1) to glycogen-enriched cell compartments and in increasing the specific activity of PP1 toward specific glycogenic substrates (glycogen synthase and glycogen phosphorylase). Here, we report a new mutation (c.746A>G, N249S) in the PPP1R3C gene that results in a decreased capacity to induce glycogen synthesis and a reduced interaction with glycogen phosphorylase and laforin, supporting a key role of this mutation in the glycogenic activity of PTG. This variant was found in one of two affected siblings of a Lafora disease family characterized by a remarkable mild course. Our findings suggest that variations in PTG may condition the course of Lafora disease and establish PTG as a potential target for pharmacogenetic and therapeutic approaches.

Lafora disease: Convergence of neurodegeneration with plant starch metabolism

Reversible phosphorylation modulates many proteins involved in glycogenesis and glycogenolysis. Recent results from mammalian cells, mouse models, eukaryotic algae, and plants have identified a new phosphorylation target in glycogen metabolism, the glucan itself.Mutations in EPM2A lead to Lafora disease, a fatal neurodegenerative disease. EPM2A encodes laforin, a bimodular protein with an amino‐terminal carbohydrate binding module and a carboxy‐terminal dual specificity phosphatase domain. We recently demonstrated that laforin removes phosphate from phosphoglucans.Starch Excess 4 (SEX4) encodes a similar phosphatase in plants, and we demonstrated that laforin and SEX4 are the founding members of the glucan phosphatases. Multiple groups have now shown that these phosphatases dephosphorylate phosphoglucans, but the physical basis for the function of laforin and SEX4 is unclear.We determined the crystal structure of SEX4, and found that it contains a phosphatase domain (DSP), carbohydrate binding module (CBM), and a previously unknown carboxy‐terminal domain. Hydrogen‐deuterium exchange mass spectrometry (DXMS) identified regions of the DSP domain that interact with glucans. Cumulatively, these results reveal the unique tertiary architecture of SEX4 that provides the physical basis for its function as a glucan phosphatase and present a model for the function of laforin.

Impaired autophagy in Lafora disease.

Lafora disease (LD) is a progressive, lethal, autosomal recessive, neurodegenerative disorder that manifests with myoclonus epilepsy. LD is characterized by the presence of intracellular inclusion bodies called Lafora bodies (LB), in brain, spinal cord and other tissues. More than 50 percent of LD is caused by mutations in EPM2A that encodes laforin. Here we review our recent findings that revealed that laforin regulates autophagy. We consider how autophagy compromise may predispose to LB formation and neurodegeneration in LD, and discuss future investigations suggested by our data.

Lafora Disease and Congenital Generalized Lipodystrophy: A Case Report

We report a patient with congenital generalized lipodystrophy who had suffered from seizures, myoclonus, ataxia and cognitive decline since late childhood. Lafora disease was diagnosed based on skin biopsy results, which revealed pathognomonic Lafora bodies. The results of genetic analysis for mutations in EPM2A and EPM2B genes were negative. This is the first case report describing an association between congenital generalized lipodystrophy and Lafora disease. Further studies focusing on the relationship between these two diseases and the identification of a third locus for Lafora disease are needed.

MR Spectroscopy Findings in Lafora Disease

Our aim was to investigate the [(1)H] MR spectroscopy (MRS) findings of Lafora Disease (LD), which is a disabling form of progressive myoclonic epilepsy. Twelve patients diagnosed with LD and 12 control subjects underwent MRS studies with single-voxels of 8 cc obtained in the frontal lobe, pons, and cerebellum. The metabolites and NAA/Cr, NAA/Cho, Cho/Cr, mI/Cr ratios were calculated. Subgroup analysis was also done between 5 patients with EPM2B and 6 patients with EPM2A mutations. Two investigators scored neurological symptom severity. We found a statistically significant difference of NAA/Cho ratio in LD patients compared with normal controls in cerebellum (P= 0.04). In addition, both myoclonus and ataxia scores showed significant correlation with NAA/Cho ratios in the pons (P= 0.03, P= 0.04) and in the cerebellum (P= 0.04, P= 0.01), respectively. We conclude that the cerebellum is the mostly affected structure in LD and there are significant correlations of MRS findings with some clinical parameters. The differences in the group may be related to different genetic mutations besides disease duration and other clinical variables. MRS studies could provide insights about the severity of the involvement of LD.

Lafora progressive myoclonus epilepsy: A meta-analysis of reported mutations in the first decade following the discovery of theEPM2AandNHLRC1genes

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.

Genetic diagnosis in Lafora disease

Lafora disease (LD) can be diagnosed by skin biopsy, but this approach has both false negatives and false positives. Biopsies of other organs can also be diagnostic but are more invasive. Genetic diagnosis is also possible but can be inconclusive, for example, in patients with only one heterozygous EPM2A mutation and patients with apparently homozygous EPM2B mutations where one parent is not a carrier of the mutation. We sought to identify occult mutations and clarify the genotypes and confirm the diagnosis of LD in patients with apparent nonrecessive disease inheritance. We used single nucleotide polymorphism, quantitative PCR, and fluorescent in situ hybridization analyses. We identified large EPM2A and EPM2B deletions undetectable by PCR in the heterozygous state and describe simple methods for their routine detection. We report a coding sequence change in several patients and describe why the pathogenic role of this change remains unclear. We confirm that adult-onset LD is due to EPM2B mutations. Finally, we report major intrafamilial heterogeneity in age at onset in LD.

Founder Effect with Variable Age at Onset in Arab Families with Lafora Disease and EPM2A Mutation

We observed three apparently unrelated and geographically separate Arab families with Lafora disease in Israel and the Palestinian territories. We clinically evaluated the families and analyzed their DNA for EPM2A mutations. Of seven individuals with Lafora disease, the clinical onset varied from 13 to 20 years. All three families shared the same novel homozygous deletion in EPM2A. Haplotype analysis around the deletion showed that the families shared a common homozygous haplotype. The boundaries of this haplotype varied between families and even within one family. We conclude that considerable variability in the age at onset of Lafora disease can occur within families. Identical mutations can be associated with the classic adolescent presentation, as well as late-onset cases. Haplotype analysis suggests that this EPM2A mutation arose many generations previously, so it may be of importance for cases distributed more widely in the Middle East.