Publications by authors named "Berge A Minassian"

151 Publications

Alleviation of a polyglucosan storage disorder by enhancement of autophagic glycogen catabolism.

EMBO Mol Med 2021 10 6;13(10):e14554. Epub 2021 Sep 6.

Laboratory for Neurodegenerative Diseases and Personalized Medicine, The Cell Screening Facility for Personalized Medicine, The Shmunis School of Biomedicine and Cancer Research, The George S. Wise Faculty for Life Sciences, Sagol School of Neurosciences, Tel Aviv University, Tel Aviv, Israel.

This work employs adult polyglucosan body disease (APBD) models to explore the efficacy and mechanism of action of the polyglucosan-reducing compound 144DG11. APBD is a glycogen storage disorder (GSD) caused by glycogen branching enzyme (GBE) deficiency causing accumulation of poorly branched glycogen inclusions called polyglucosans. 144DG11 improved survival and motor parameters in a GBE knockin (Gbe ) APBD mouse model. 144DG11 reduced polyglucosan and glycogen in brain, liver, heart, and peripheral nerve. Indirect calorimetry experiments revealed that 144DG11 increases carbohydrate burn at the expense of fat burn, suggesting metabolic mobilization of pathogenic polyglucosan. At the cellular level, 144DG11 increased glycolytic, mitochondrial, and total ATP production. The molecular target of 144DG11 is the lysosomal membrane protein LAMP1, whose interaction with the compound, similar to LAMP1 knockdown, enhanced autolysosomal degradation of glycogen and lysosomal acidification. 144DG11 also enhanced mitochondrial activity and modulated lysosomal features as revealed by bioenergetic, image-based phenotyping and proteomics analyses. As an effective lysosomal targeting therapy in a GSD model, 144DG11 could be developed into a safe and efficacious glycogen and lysosomal storage disease therapy.
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http://dx.doi.org/10.15252/emmm.202114554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8495453PMC
October 2021

Retinal alterations in patients with Lafora disease.

Am J Ophthalmol Case Rep 2021 Sep 15;23:101146. Epub 2021 Jun 15.

Division of Neurology, Department of Pediatrics, University of Texas Southwestern, Dallas, TX, USA.

Purpose: Lafora disease is a genetic neurodegenerative metabolic disorder caused by insoluble polyglucosan aggregate accumulation throughout the central nervous system and body. The retina is an accessible neural tissue, which may offer alternative methods to assess neurological diseases quickly and noninvasively. In this way, noninvasive imaging may provide a means to characterize neurodegenerative disease, which enables earlier identification and diagnosis of disease and the ability to monitor disease progression. In this study, we sought to characterize the retina of individuals with Lafora disease using non-invasive retinal imaging.

Methods: One eye of three individuals with genetically confirmed Lafora disease were imaged with optical coherence tomography (OCT) and adaptive optics scanning light ophthalmoscopy (AOSLO). When possible, OCT volume and line scans were acquired to assess total retinal thickness, ganglion cell-inner plexiform layer thickness, and outer nuclear layer + Henle fiber layer thickness. OCT angiography (OCTA) scans were acquired in one subject at the macula and optic nerve head (ONH). AOSLO was used to characterize the photoreceptor mosaic and examine the retinal nerve fiber layer (RNFL).

Results: Two subjects with previous seizure activity demonstrated reduced retinal thickness, while one subject with no apparent symptoms had normal retinal thickness. All other clinical measures, as well as parafoveal cone density, were within normal range. Nummular reflectivity at the level of the RNFL was observed using AOSLO in the macula and near the ONH in all three subjects.

Conclusions: This multimodal retinal imaging approach allowed us to observe a number of retinal structural features in all three individuals. Most notably, AOSLO revealed nummular reflectivity within the inner retina of each subject. This phenotype has not been reported previously and may represent a characteristic change produced by the neurodegenerative process.
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http://dx.doi.org/10.1016/j.ajoc.2021.101146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8239732PMC
September 2021

EPM2A in-frame deletion slows neurological decline in Lafora Disease.

Seizure 2021 Oct 10;91:97-98. Epub 2021 Jun 10.

Department of Pediatrics-Neurology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, Texas 75390-9063, United States. Electronic address:

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http://dx.doi.org/10.1016/j.seizure.2021.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8434975PMC
October 2021

Gys1 antisense therapy rescues neuropathological bases of murine Lafora disease.

Brain 2021 May 16. Epub 2021 May 16.

Program in Genetics and Genome Biology, The Hospital for Sick Children Research Institute, Toronto, ON M5G 0A4, Canada.

Lafora disease is a fatal progressive myoclonus epilepsy. At root, it is due to constant acquisition of branches that are too long in a subgroup of glycogen molecules, leading them to precipitate and accumulate into Lafora bodies, which drive a neuroinflammatory response and neurodegeneration. As a potential therapy, we aimed to downregulate glycogen synthase, the enzyme responsible for glycogen branch elongation, in the disease's mouse models. We synthesized an antisense oligonucleotide (Gys1-ASO) that targets the mRNA of the brain-expressed glycogen synthase 1 gene (Gys1). We administered Gys1-ASO by intracerebroventricular injection and analyzed the pathological hallmarks of Lafora disease, namely glycogen accumulation, Lafora body formation, and neuroinflammation. Gys1-ASO prevented Lafora body formation in young mice that had not yet formed them. In older mice that already exhibited Lafora bodies, Gys1-ASO inhibited further accumulation, markedly preventing large Lafora bodies characteristic of advanced disease. Inhibition of Lafora body formation was associated with prevention of astrogliosis and strong trends towards correction of dysregulated expression of disease immune and neuroinflammatory markers. Lafora disease manifests gradually in previously healthy teenagers. Our work provides proof of principle that an antisense oligonucleotide targeting the GYS1 mRNA could prevent, and halt progression of, this catastrophic epilepsy.
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http://dx.doi.org/10.1093/brain/awab194DOI Listing
May 2021

Targeting Gys1 with AAV-SaCas9 Decreases Pathogenic Polyglucosan Bodies and Neuroinflammation in Adult Polyglucosan Body and Lafora Disease Mouse Models.

Neurotherapeutics 2021 04 8;18(2):1414-1425. Epub 2021 Apr 8.

Division of Neurology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.

Many adult and most childhood neurological diseases have a genetic basis. CRISPR/Cas9 biotechnology holds great promise in neurological therapy, pending the clearance of major delivery, efficiency, and specificity hurdles. We applied CRISPR/Cas9 genome editing in its simplest modality, namely inducing gene sequence disruption, to one adult and one pediatric disease. Adult polyglucosan body disease is a neurodegenerative disease resembling amyotrophic lateral sclerosis. Lafora disease is a severe late childhood onset progressive myoclonus epilepsy. The pathogenic insult in both is formation in the brain of glycogen with overlong branches, which precipitates and accumulates into polyglucosan bodies that drive neuroinflammation and neurodegeneration. We packaged Staphylococcus aureus Cas9 and a guide RNA targeting the glycogen synthase gene, Gys1, responsible for brain glycogen branch elongation in AAV9 virus, which we delivered by neonatal intracerebroventricular injection to one mouse model of adult polyglucosan body disease and two mouse models of Lafora disease. This resulted, in all three models, in editing of approximately 17% of Gys1 alleles and a similar extent of reduction of Gys1 mRNA across the brain. The latter led to approximately 50% reductions of GYS1 protein, abnormal glycogen accumulation, and polyglucosan bodies, as well as ameliorations of neuroinflammatory markers in all three models. Our work represents proof of principle for virally delivered CRISPR/Cas9 neurotherapeutics in an adult-onset (adult polyglucosan body) and a childhood-onset (Lafora) neurological diseases.
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http://dx.doi.org/10.1007/s13311-021-01040-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8423949PMC
April 2021

The antioxidant MnTBAP does not effectively downregulate CD4 expression in T cells in vivo.

J Neuroimmunol 2021 05 8;354:577544. Epub 2021 Mar 8.

Department of Neurology & Neurotherapeutics, University of Texas Southwestern Medical School, Dallas, TX, USA; Neurology Section, VA North Texas Health Care System, Dallas, TX, USA. Electronic address:

The antioxidant MnTBAP was previously shown to down-regulate the surface expression of CD4 molecule in T cells. This observation obviously holds great potential impact in a number of pathological human conditions, including autoimmunity. Three different single doses of MnTBAP reduced the frequency of CD4 cells. However, the median florescent intensity (MFI) was not different. Initiation of in vivo pharmacotherapy or vehicle control was performed inC57BL/6 mice that were actively immunized for experimental autoimmune encephalomyelitis (EAE). In contrast to published reports, the mean frequency of CD4 cells, and the median fluorescent intensity (MFI) of CD4 was similar in both treatment groups. 25-day survival following active immunization among the MnTBAP treated animals compared to vehicle controls was16.6 ± 6.9 days vs 23.6 ± 2.7 days; (P value <0.05). We conclude that MnTBAP (Sack and Herzog, 2009 (Sack and Herzog, 2009)) does not effectively downregulate CD4 expression in T cells in vivo, probably due to extensive mechanism that distinguishes it from an in vitro model (Harding, 1993 (Harding, 1993)) possesses toxic properties that may limit its clinic use in possible doses that could deliver the immunomodulation through down regulation of CD4 expression, and (Saizawa et al., 1987 (Saizawa et al., 1987)) has limited availability in specific tissues, including the CNS.
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http://dx.doi.org/10.1016/j.jneuroim.2021.577544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8109275PMC
May 2021

Ketogenic diet reduces Lafora bodies in murine Lafora disease.

Neurol Genet 2020 Dec 19;6(6):e533. Epub 2020 Nov 19.

Institute of Medical Science (L.I.), University of Toronto, ON; Program in Genetics and Genome Biology (L.I., P.W., S.G., X.Z., B.A.M.), The Hospital for Sick Children Research Institute, Toronto, ON, Canada; and Division of Neurology (B.A.M.), Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas.

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http://dx.doi.org/10.1212/NXG.0000000000000533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7713716PMC
December 2020

Exploiting the diphtheria toxin internalization receptor enhances delivery of proteins to lysosomes for enzyme replacement therapy.

Sci Adv 2020 12 11;6(50). Epub 2020 Dec 11.

Molecular Medicine Program, The Hospital for Sick Children, Toronto, ON, Canada.

Enzyme replacement therapy, in which a functional copy of an enzyme is injected either systemically or directly into the brain of affected individuals, has proven to be an effective strategy for treating certain lysosomal storage diseases. The inefficient uptake of recombinant enzymes via the mannose-6-phosphate receptor, however, prohibits the broad utility of replacement therapy. Here, to improve the efficiency and efficacy of lysosomal enzyme uptake, we exploited the strategy used by diphtheria toxin to enter into the endolysosomal network of cells by creating a chimera between the receptor-binding fragment of diphtheria toxin and the lysosomal hydrolase TPP1. We show that chimeric TPP1 binds with high affinity to target cells and is efficiently delivered into lysosomes. Further, we show superior uptake of chimeric TPP1 over TPP1 alone in brain tissue following intracerebroventricular injection in mice lacking TPP1, demonstrating the potential of this strategy for enhancing lysosomal storage disease therapy.
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http://dx.doi.org/10.1126/sciadv.abb0385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7732195PMC
December 2020

An inducible glycogen synthase-1 knockout halts but does not reverse Lafora disease progression in mice.

J Biol Chem 2021 Jan-Jun;296:100150. Epub 2020 Dec 10.

Division of Neurology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas, USA; Program in Genetics and Genome Biology, The Hospital for Sick Children Research Institute, Toronto, Ontario, Canada. Electronic address:

Malstructured glycogen accumulates over time in Lafora disease (LD) and precipitates into Lafora bodies (LBs), leading to neurodegeneration and intractable fatal epilepsy. Constitutive reduction of glycogen synthase-1 (GYS1) activity prevents murine LD, but the effect of GYS1 reduction later in disease course is unknown. Our goal was to knock out Gys1 in laforin (Epm2a)-deficient LD mice after disease onset to determine whether LD can be halted in midcourse, or even reversed. We generated Epm2a-deficient LD mice with tamoxifen-inducible Cre-mediated Gys1 knockout. Tamoxifen was administered at 4 months and disease progression assessed at 12 months. We verified successful knockout at mRNA and protein levels using droplet digital PCR and Western blots. Glycogen determination and periodic acid-Schiff-diastase staining were used to analyze glycogen and LB accumulation. Immunohistochemistry using astrocytic (glial fibrillary acidic protein) and microglial (ionized calcium-binding adapter molecule 1) markers was performed to investigate neuroinflammation. In the disease-relevant organ, the brain, Gys1 mRNA levels were reduced by 85% and GYS1 protein depleted. Glycogen accumulation was halted at the 4-month level, while LB formation and neuroinflammation were significantly, though incompletely, prevented. Skeletal muscle analysis confirmed that Gys1 knockout inhibits glycogen and LB accumulation. However, tamoxifen-independent Cre recombination precluded determination of disease halting or reversal in this tissue. Our study shows that Gys1 knockdown is a powerful means to prevent LD progression, but this approach did not reduce brain glycogen or LBs to levels below those at the time of intervention. These data suggest that endogenous mechanisms to clear brain LBs are absent or, possibly, compromised in laforin-deficient murine LD.
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http://dx.doi.org/10.1074/jbc.RA120.015773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857511PMC
August 2021

SUCLA2 Arg407Trp mutation can cause a nonprogressive movement disorder - deafness syndrome.

Ann Clin Transl Neurol 2021 01 24;8(1):252-258. Epub 2020 Nov 24.

Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, Ontario, Canada.

SUCLA2 is a component of mitochondrial succinate-CoA ligase and nucleotide diphosphokinase activities. Its absence results in Krebs cycle failure, mitochondrial DNA depletion, and a childhood-fatal encephalomyopathy. We describe a purely neurologic allelic form of the disease consisting of deafness, putamenal hyperintensity on MRI and a myoclonic-dystonic movement disorder unchanging from childhood into, so far, the late fourth decade. We show that succinate supplementation circumvents the Krebs cycle block, but does not correct the neurologic disease. Our patients' Arg407Trp mutation has been reported in children with (yet) no MRI abnormalities. It remains possible that early succinate supplementation could impact the disease.
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http://dx.doi.org/10.1002/acn3.51247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7818133PMC
January 2021

SUCLA2 mutations cause global protein succinylation contributing to the pathomechanism of a hereditary mitochondrial disease.

Nat Commun 2020 11 23;11(1):5927. Epub 2020 Nov 23.

Gladstone Institutes and University of California, San Francisco, CA, 94158, USA.

Mitochondrial acyl-coenzyme A species are emerging as important sources of protein modification and damage. Succinyl-CoA ligase (SCL) deficiency causes a mitochondrial encephalomyopathy of unknown pathomechanism. Here, we show that succinyl-CoA accumulates in cells derived from patients with recessive mutations in the tricarboxylic acid cycle (TCA) gene succinyl-CoA ligase subunit-β (SUCLA2), causing global protein hyper-succinylation. Using mass spectrometry, we quantify nearly 1,000 protein succinylation sites on 366 proteins from patient-derived fibroblasts and myotubes. Interestingly, hyper-succinylated proteins are distributed across cellular compartments, and many are known targets of the (NAD)-dependent desuccinylase SIRT5. To test the contribution of hyper-succinylation to disease progression, we develop a zebrafish model of the SCL deficiency and find that SIRT5 gain-of-function reduces global protein succinylation and improves survival. Thus, increased succinyl-CoA levels contribute to the pathology of SCL deficiency through post-translational modifications.
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http://dx.doi.org/10.1038/s41467-020-19743-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7684291PMC
November 2020

GYS1 or PPP1R3C deficiency rescues murine adult polyglucosan body disease.

Ann Clin Transl Neurol 2020 11 9;7(11):2186-2198. Epub 2020 Oct 9.

Genetics and Genome Biology Program, The Hospital for Sick Children, Peter Gilgan Centre for Research and Learning, Toronto, Ontario, Canada.

Objective: Adult polyglucosan body disease (APBD) is an adult-onset neurological variant of glycogen storage disease type IV. APBD is caused by recessive mutations in the glycogen branching enzyme gene, and the consequent accumulation of poorly branched glycogen aggregates called polyglucosan bodies in the nervous system. There are presently no treatments for APBD. Here, we test whether downregulation of glycogen synthesis is therapeutic in a mouse model of the disease.

Methods: We characterized the effects of knocking out two pro-glycogenic proteins in an APBD mouse model. APBD mice were crossed with mice deficient in glycogen synthase (GYS1), or mice deficient in protein phosphatase 1 regulatory subunit 3C (PPP1R3C), a protein involved in the activation of GYS1. Phenotypic and histological parameters were analyzed and glycogen was quantified.

Results: APBD mice deficient in GYS1 or PPP1R3C demonstrated improvements in life span, morphology, and behavioral assays of neuromuscular function. Histological analysis revealed a reduction in polyglucosan body accumulation and of astro- and micro-gliosis in the brains of GYS1- and PPP1R3C-deficient APBD mice. Brain glycogen quantification confirmed the reduction in abnormal glycogen accumulation. Analysis of skeletal muscle, heart, and liver found that GYS1 deficiency reduced polyglucosan body accumulation in all three tissues and PPP1R3C knockout reduced skeletal muscle polyglucosan bodies.

Interpretation: GYS1 and PPP1R3C are effective therapeutic targets in the APBD mouse model. These findings represent a critical step toward the development of a treatment for APBD and potentially other glycogen storage disease type IV patients.
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http://dx.doi.org/10.1002/acn3.51211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7664254PMC
November 2020

Ppp1r3d deficiency preferentially inhibits neuronal and cardiac Lafora body formation in a mouse model of the fatal epilepsy Lafora disease.

J Neurochem 2021 06 10;157(6):1897-1910. Epub 2020 Oct 10.

Institute of Medical Science, University of Toronto, Toronto, ON, Canada.

Mammalian glycogen chain lengths are subject to complex regulation, including by seven proteins (protein phosphatase-1 regulatory subunit 3, PPP1R3A through PPP1R3G) that target protein phosphatase-1 (PP1) to glycogen to activate the glycogen chain-elongating enzyme glycogen synthase and inactivate the chain-shortening glycogen phosphorylase. Lafora disease is a fatal neurodegenerative epilepsy caused by aggregates of long-chained, and as a result insoluble, glycogen, termed Lafora bodies (LBs). We previously eliminated PPP1R3C from a Lafora disease mouse model and studied the effect on LB formation. In the present work, we eliminate and study the effect of absent PPP1R3D. In the interim, brain cell type levels of all PPP1R3 genes have been published, and brain cell type localization of LBs clarified. Integrating these data we find that PPP1R3C is the major isoform in most tissues including brain. In the brain, PPP1R3C is expressed at 15-fold higher levels than PPP1R3D in astrocytes, the cell type where most LBs form. PPP1R3C deficiency eliminates ~90% of brain LBs. PPP1R3D is quantitatively a minor isoform, but possesses unique MAPK, CaMK2 and 14-3-3 binding domains and appears to have an important functional niche in murine neurons and cardiomyocytes. In neurons, it is expressed equally to PPP1R3C, and its deficiency eliminates ~50% of neuronal LBs. In heart, it is expressed at 25% of PPP1R3C where its deficiency eliminates ~90% of LBs. This work studies the role of a second (PPP1R3D) of seven PP1 subunits that regulate the structure of glycogen, toward better understanding of brain glycogen metabolism generally, and in Lafora disease.
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http://dx.doi.org/10.1111/jnc.15176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7936008PMC
June 2021

Sensitive quantification of α-glucans in mouse tissues, cell cultures, and human cerebrospinal fluid.

J Biol Chem 2020 10 13;295(43):14698-14709. Epub 2020 Aug 13.

Departments of Pediatrics and Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA. Electronic address:

The soluble α-polyglucan glycogen is a central metabolite enabling transient glucose storage to suit cellular energy needs. Glycogen storage diseases (GSDs) comprise over 15 entities caused by generalized or tissue-specific defects in enzymes of glycogen metabolism. In several, in Lafora disease caused by the absence of the glycogen phosphatase laforin or its interacting partner malin, degradation-resistant abnormally structured insoluble glycogen accumulates. Sensitive quantification methods for soluble and insoluble glycogen are critical to research, including therapeutic studies, in such diseases. This paper establishes methodological advancements relevant to glycogen metabolism investigations generally, and GSDs. Introducing a pre-extraction incubation method, we measure degradation-resistant glycogen in as little as 30 mg of skeletal muscle or a single hippocampus from Lafora disease mouse models. The digestion-resistant glycogen correlates with the disease-pathogenic insoluble glycogen and can readily be detected in very young mice where glycogen accumulation has just begun. Second, we establish a high-sensitivity glucose assay with detection of ATP depletion, enabling 1) quantification of α-glucans in cell culture using a medium-throughput assay suitable for assessment of candidate glycogen synthesis inhibitors, and 2) discovery of α-glucan material in healthy human cerebrospinal fluid, establishing a novel methodological platform for biomarker analyses in Lafora disease and other GSDs.
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http://dx.doi.org/10.1074/jbc.RA120.015061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7586225PMC
October 2020

Defining the phenotype of FHF1 developmental and epileptic encephalopathy.

Epilepsia 2020 07 9;61(7):e71-e78. Epub 2020 Jul 9.

Pediatric Neurology, University Hospitals Leuven, Leuven, Belgium.

Fibroblast growth-factor homologous factor (FHF1) gene variants have recently been associated with developmental and epileptic encephalopathy (DEE). FHF1 encodes a cytosolic protein that modulates neuronal sodium channel gating. We aim to refine the electroclinical phenotypic spectrum of patients with pathogenic FHF1 variants. We retrospectively collected clinical, genetic, neurophysiologic, and neuroimaging data of 17 patients with FHF1-DEE. Sixteen patients had recurrent heterozygous FHF1 missense variants: 14 had the recurrent p.Arg114His variant and two had a novel likely pathogenic variant p.Gly112Ser. The p.Arg114His variant is associated with an earlier onset and more severe phenotype. One patient carried a chromosomal microduplication involving FHF1. Twelve patients carried a de novo variant, five (29.5%) inherited from parents with gonadic or somatic mosaicism. Seizure onset was between 1 day and 41 months; in 76.5% it was within 30 days. Tonic seizures were the most frequent seizure type. Twelve patients (70.6%) had drug-resistant epilepsy, 14 (82.3%) intellectual disability, and 11 (64.7%) behavioral disturbances. Brain magnetic resonance imaging (MRI) showed mild cerebral and/or cerebellar atrophy in nine patients (52.9%). Overall, our findings expand and refine the clinical, EEG, and imaging phenotype of patients with FHF1-DEE, which is characterized by early onset epilepsy with tonic seizures, associated with moderate to severe ID and psychiatric features.
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http://dx.doi.org/10.1111/epi.16582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8168379PMC
July 2020

Epilepsy phenotype in individuals with chromosomal duplication encompassing .

Epilepsia Open 2020 Jun 9;5(2):301-306. Epub 2020 May 9.

Program in Genetics and Genome Biology Hospital for Sick Children Research Institute Toronto ON Canada.

Intragenic mutations in are associated with intractable seizures, developmental regression, intellectual disability, ataxia, hypotonia, and feeding difficulties. duplications are rarely reported, but it was suggested that those might have a similar gain-of-function effect and lead to a more or less comparable phenotype. A favorable response to the sodium blocker phenytoin was reported in several cases, both in patients with an intragenic mutation and in patients with a duplication of . We report three individuals from two families with duplications. The duplications are flanked and probably mediated by two long interspersed nuclear elements (LINEs). The duplication cases show phenotypic overlap with the cases with intragenic mutations. Though the onset of epilepsy might be later, after the onset of seizures both groups show developmental stagnation and regression in several cases. This illustrates and further confirms that chromosomal duplications and intragenic gain-of-function mutations yield overlapping phenotypes.
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http://dx.doi.org/10.1002/epi4.12396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278552PMC
June 2020

From Genetic Testing to Precision Medicine in Epilepsy.

Neurotherapeutics 2020 04;17(2):609-615

Department of Pediatrics Division of Neurology, University of Texas Southwestern, Dallas, Texas, USA.

Epilepsy includes a number of medical conditions with recurrent seizures as common denominator. The large number of different syndromes and seizure types as well as the highly variable inter-individual response to the therapies makes management of this condition often challenging. In the last two decades, a genetic etiology has been revealed in more than half of all epilepsies and single gene defects in ion channels or neurotransmitter receptors have been associated with most inherited forms of epilepsy, including some focal and lesional forms as well as specific epileptic developmental encephalopathies. Several genetic tests are now available, including targeted assays up to revolutionary tools that have made sequencing of all coding (whole exome) and non-coding (whole genome) regions of the human genome possible. These recent technological advances have also driven genetic discovery in epilepsy and increased our understanding of the molecular mechanisms of many epileptic disorders, eventually providing targets for precision medicine in some syndromes, such as Dravet syndrome, pyroxidine-dependent epilepsy, and glucose transporter 1 deficiency. However, these examples represent a relatively small subset of all types of epilepsy, and to date, precision medicine in epilepsy has primarily focused on seizure control, and other clinical aspects, such as neurodevelopmental and neuropsychiatric comorbidities, have yet been possible to address. We herein summarize the most recent advances in genetic testing and provide up-to-date approaches for the choice of the correct test for some epileptic disorders and tailored treatments that are already applicable in some monogenic epilepsies. In the next years, the most probably scenario is that epilepsy treatment will be very different from the currently almost empirical approach, eventually with a "precision medicine" approach applicable on a large scale.
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http://dx.doi.org/10.1007/s13311-020-00835-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7283411PMC
April 2020

The 5th International Lafora Epilepsy Workshop: Basic science elucidating therapeutic options and preparing for therapies in the clinic.

Epilepsy Behav 2020 02 10;103(Pt A):106839. Epub 2020 Jan 10.

Lafora Epilepsy Cure Initiative (LECI), USA; Laboratory of Neurology, IIS-Jimenez Diaz Foundation, UAM, 28045 Madrid, Spain; Biomedical Research Networking Center on Rare Diseases (CIBERER), 28029 Madrid, Spain.

Lafora disease (LD) is both a fatal childhood epilepsy and a glycogen storage disease caused by recessive mutations in either the Epilepsy progressive myoclonus 2A (EPM2A) or EPM2B genes. Hallmarks of LD are aberrant, cytoplasmic carbohydrate aggregates called Lafora bodies (LBs) that are a disease driver. The 5th International Lafora Epilepsy Workshop was recently held in Alcala de Henares, Spain. The workshop brought together nearly 100 clinicians, academic and industry scientists, trainees, National Institutes of Health (NIH) representation, and friends and family members of patients with LD. The workshop covered aspects of LD ranging from defining basic scientific mechanisms to elucidating a LD therapy or cure and a recently launched LD natural history study.
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http://dx.doi.org/10.1016/j.yebeh.2019.106839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7024738PMC
February 2020

Sexually Divergent Mortality and Partial Phenotypic Rescue After Gene Therapy in a Mouse Model of Dravet Syndrome.

Hum Gene Ther 2020 03 16;31(5-6):339-351. Epub 2020 Jan 16.

Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Canada.

Dravet syndrome (DS) is a neurodevelopmental genetic disorder caused by mutations in the gene encoding the α subunit of the NaV1.1 voltage-gated sodium channel that controls neuronal action potential firing. The high density of this mutated channel in GABAergic interneurons results in impaired inhibitory neurotransmission and subsequent excessive activation of excitatory neurons. The syndrome is associated with severe childhood epilepsy, autistic behaviors, and sudden unexpected death in epilepsy. Here, we compared the rescue effects of an adeno-associated viral (AAV) vector coding for the multifunctional β1 sodium channel auxiliary subunit (AAV-NaVβ1) with a control vector lacking a transgene. We hypothesized that overexpression of NaVβ1 would facilitate the function of residual voltage-gated channels and improve the DS phenotype in the mouse model of DS. AAV-NaVβ1 was injected into the cerebral spinal fluid of neonatal mice. In untreated control mice, females showed a higher degree of mortality than males. Compared with control mice, AAV-NaVβ1-treated mice displayed increased survival, an outcome that was more pronounced in females than males. In contrast, behavioral analysis revealed that male, but not female, mice displayed motor hyperactivity, and abnormal performance on tests of fear and anxiety and learning and memory. Male mice treated with AAV-NaVβ1 showed reduced spontaneous seizures and normalization of motor activity and performance on the elevated plus maze test. These findings demonstrate sex differences in mortality in untreated mice, an effect that may be related to a lower level of intrinsic inhibitory tone in female mice, and a normalization of aberrant behaviors in males after central nervous system administration of AAV-NaVβ1. The therapeutic efficacy of AAV-NaVβ1 in a mouse model of DS suggests a potential new long-lasting biological therapeutic avenue for the treatment of this catastrophic epilepsy.
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http://dx.doi.org/10.1089/hum.2019.225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7087406PMC
March 2020

Re-annotation of 191 developmental and epileptic encephalopathy-associated genes unmasks de novo variants in .

NPJ Genom Med 2019 2;4:31. Epub 2019 Dec 2.

20Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, CB2 0XY UK.

The developmental and epileptic encephalopathies (DEE) are a group of rare, severe neurodevelopmental disorders, where even the most thorough sequencing studies leave 60-65% of patients without a molecular diagnosis. Here, we explore the incompleteness of transcript models used for exome and genome analysis as one potential explanation for a lack of current diagnoses. Therefore, we have updated the GENCODE gene annotation for 191 epilepsy-associated genes, using human brain-derived transcriptomic libraries and other data to build 3,550 putative transcript models. Our annotations increase the transcriptional 'footprint' of these genes by over 674 kb. Using as a case study, due to its close phenotype/genotype correlation with Dravet syndrome, we screened 122 people with Dravet syndrome or a similar phenotype with a panel of exon sequences representing eight established genes and identified two de novo variants that now - through improved gene annotation - are ascribed to residing among our exons. These two (from 122 screened people, 1.6%) molecular diagnoses carry significant clinical implications. Furthermore, we identified a previously classified intronic Dravet syndrome-associated variant that now lies within a deeply conserved exon. Our findings illustrate the potential gains of thorough gene annotation in improving diagnostic yields for genetic disorders.
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http://dx.doi.org/10.1038/s41525-019-0106-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6889285PMC
December 2019

The best evidence for progressive myoclonic epilepsy: A pathway to precision therapy.

Seizure 2019 Oct 23;71:247-257. Epub 2019 Aug 23.

Pediatric Neurology and Muscular Diseases Unit, IRCCS Istituto 'G. Gaslini', Italy; Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genova, Genova, Italy.

Progressive Myoclonus Epilepsies (PMEs) are a group of uncommon clinically and genetically heterogeneous disorders characterised by myoclonus, generalized epilepsy, and neurological deterioration, including dementia and ataxia. PMEs may have infancy, childhood, juvenile or adult onset, but usually present in late childhood or adolescence, at variance from epileptic encephalopathies, which start with polymorphic seizures in early infancy. Neurophysiologic recordings are suited to describe faithfully the time course of the shock-like muscle contractions which characterize myoclonus. A combination of positive and negative myoclonus is typical of PMEs. The gene defects for most PMEs (Unverricht-Lundborg disease, Lafora disease, several forms of neuronal ceroid lipofuscinoses, myoclonus epilepsy with ragged-red fibers [MERRF], and type 1 and 2 sialidoses) have been identified. PMEs are uncommon disorders, difficult to diagnose in the absence of extensive experience. Thus, aetiology is undetermined in many patients, despite the advance in molecular medicine. Treatment of PMEs remains essentially symptomaticof seizures and myoclonus, together with palliative, supportive, and rehabilitative measures. The response to therapy may initially be relatively favourable, afterwards however, seizures may become more frequent, and progressive neurologic decline occurs. The prognosis of a PME depends on the specific disease. The history of PMEs revealed that the international collaboration and sharing experience is the right way to proceed. This emerging picture and biological insights will allow us to find ways to provide the patients with meaningful treatment.
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http://dx.doi.org/10.1016/j.seizure.2019.08.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7288863PMC
October 2019

Both gain-of-function and loss-of-function de novo CACNA1A mutations cause severe developmental epileptic encephalopathies in the spectrum of Lennox-Gastaut syndrome.

Epilepsia 2019 09 29;60(9):1881-1894. Epub 2019 Aug 29.

Sainte-Justine University Hospital Center, University of Montréal, Montréal, Canada.

Objective: Developmental epileptic encephalopathies (DEEs) are genetically heterogeneous severe childhood-onset epilepsies with developmental delay or cognitive deficits. In this study, we explored the pathogenic mechanisms of DEE-associated de novo mutations in the CACNA1A gene.

Methods: We studied the functional impact of four de novo DEE-associated CACNA1A mutations, including the previously described p.A713T variant and three novel variants (p.V1396M, p.G230V, and p.I1357S). Mutant cDNAs were expressed in HEK293 cells, and whole-cell voltage-clamp recordings were conducted to test the impacts on Ca 2.1 channel function. Channel localization and structure were assessed with immunofluorescence microscopy and three-dimensional (3D) modeling.

Results: We find that the G230V and I1357S mutations result in loss-of-function effects with reduced whole-cell current densities and decreased channel expression at the cell membrane. By contrast, the A713T and V1396M variants resulted in gain-of-function effects with increased whole-cell currents and facilitated current activation (hyperpolarized shift). The A713T variant also resulted in slower current decay. 3D modeling predicts conformational changes favoring channel opening for A713T and V1396M.

Significance: Our findings suggest that both gain-of-function and loss-of-function CACNA1A mutations are associated with similarly severe DEEs and that functional validation is required to clarify the underlying molecular mechanisms and to guide therapies.
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http://dx.doi.org/10.1111/epi.16316DOI Listing
September 2019

Skeletal Muscle Glycogen Chain Length Correlates with Insolubility in Mouse Models of Polyglucosan-Associated Neurodegenerative Diseases.

Cell Rep 2019 04;27(5):1334-1344.e6

Program in Genetics and Genome Biology, Hospital for Sick Children Research Institute, Toronto, ON M5G 0A4, Canada. Electronic address:

Lafora disease (LD) and adult polyglucosan body disease (APBD) are glycogen storage diseases characterized by a pathogenic buildup of insoluble glycogen. Mechanisms causing glycogen insolubility are poorly understood. Here, in two mouse models of LD (Epm2a and Epm2b) and one of APBD (Gbe1), the separation of soluble and insoluble muscle glycogen is described, enabling separate analysis of each fraction. Total glycogen is increased in LD and APBD mice, which, together with abnormal chain length and molecule size distributions, is largely if not fully attributed to insoluble glycogen. Soluble glycogen consists of molecules with distinct chain length distributions and differential corresponding solubility, providing a mechanistic link between soluble and insoluble glycogen in vivo. Phosphorylation states differ across glycogen fractions and mouse models, demonstrating that hyperphosphorylation is not a basic feature of insoluble glycogen. Lastly, model-specific variances in protein and activity levels of key glycogen synthesis enzymes suggest uninvestigated regulatory mechanisms.
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http://dx.doi.org/10.1016/j.celrep.2019.04.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6530600PMC
April 2019

Dominant mutation causes intellectual disability with remitting epilepsy.

Ann Clin Transl Neurol 2019 Apr 7;6(4):807-811. Epub 2019 Mar 7.

Program in Genetics and Genome Biology The Hospital for Sick Children Toronto Ontario Canada.

Mis-secreted glycoproteins (LGI1, reelin) are emerging causes of epilepsy. LMAN2L belongs to a glycoprotein secretion chaperone family. One recessive missense mutation predicted to impair the chaperone's interaction with glycoproteins was reported in a family with intellectual disability (ID) and remitting epilepsy. We describe four members of a family with autosomal dominant inheritance of a similar phenotype. We show that they segregate a NM_001142292.1:c.1073delT mutation that eliminates LMAN2L's endoplasmic reticulum retention signal and mislocalizes the protein from that compartment to the plasma membrane. LMAN2L mislocalization, like impaired glycoprotein interaction, disturbs brain development, including generation of developmentally restricted epilepsy.
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http://dx.doi.org/10.1002/acn3.727DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6469342PMC
April 2019

EEG of asymptomatic first-degree relatives of patients with juvenile myoclonic, childhood absence and rolandic epilepsy: a systematic review and meta-analysis.

Epileptic Disord 2019 Feb;21(1):30-41

Program in Genetics and Genome Biology, The Hospital for Sick Children, Institute of Medical Science, University of Toronto, Toronto, Division of Neurology, Department of Pediatrics, University of Texas Southwestern, Dallas, USA.

Rolandic (RE), childhood absence (CAE) and juvenile myoclonic (JME) epilepsy encompass centrotemporal sharp waves, 3-Hz spike waves and >3-Hz spike or polyspike waves, respectively. Evidence abounds for genetic roles in all three syndromes, yet involved genes for the vast majority of patients remain unknown. It has long been proposed that while each disease is genetically complex, its specific EEG trait may represent a genetically simpler endophenotype. This meta-analysis of the literature focuses on the frequency of EEG traits in clinically unaffected first-degree relatives towards determining inheritance patterns of the EEG endophenotypes. We used the Preferred Reporting Items for Systematic Review and Meta-Analysis for protocols (PRISMA-P) and searched Medline, EMBASE, CINHAL and the Cochrane Central Register of Controlled Trials. Following extensive screening, 15 studies were included with a total of 3,858 asymptomatic relatives. The prevalence of 'abnormal' EEG waves was 21%, 42% and 33% for JME, CAE and RE, respectively, close to what would be expected based on Mendelian inheritance. However, breaking down the reported EEG abnormalities, most consisted not of the respective EEG signature traits -prevalences of which were as low as 5%- but of non-specific EEG 'abnormalities'/variants. Prevalence of non-specific EEG 'abnormalities'/variants in the general population ranges from 0.1 to 10%. Underlying this 100-fold-wide range is a spectrum of what is considered 'abnormal' or variant. The prevalences of 'abnormalities'/variants in asymptomatic siblings in RE, CAE and JME significantly exceed even the highest value in the general population and fall within Mendelian expectations. These results suggest that EEG 'abnormalities'/variants shared with the general population are enriched in the three syndromes and are endophenotypes inherited in a genetically simple near-Mendelian fashion. Future work with modern EEG variant definitions should uncover genetic variants contributing to neuronal hypersynchrony in epilepsy.
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http://dx.doi.org/10.1684/epd.2019.1024DOI Listing
February 2019

Diabetes Mellitus in a Patient With Lafora Disease: Possible Links With Pancreatic β-Cell Dysfunction and Insulin Resistance.

Front Pediatr 2018 16;6:424. Epub 2019 Jan 16.

Division of Endocrinology, Department of Pediatrics, Sidra Medicine Outpatient Clinic, Doha, Qatar.

Lafora disease (LD) is a rare autosomal recessive disorder characterized by progressive myoclonic epilepsy followed by continuous neurological decline, culminating in death within 10 years. LD leads to accumulation of insoluble, abnormal, glycogen-like structures called Lafora bodies (LBs). It is caused by mutations in the gene encoding glycogen phosphatase ( or the E3 ubiquitin ligase malin (. These two proteins are involved in an intricate, however, incompletely elucidated pathway governing glycogen metabolism. The formation of EPM2A and malin signaling complex promotes the ubiquitination of proteins participating in glycogen metabolism, where dysfunctional mutations lead to the formation of LBs. Herein, we describe a 13-years-old child with LD due to a (c.386C > A, p.Pro129His) mutation, who has developed diabetes mellitus and was treated with metformin. We discuss how basic mechanisms of LD could be linked to β-cell dysfunction and insulin resistance.
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http://dx.doi.org/10.3389/fped.2018.00424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343460PMC
January 2019

is associated with recessive primary familial brain calcification.

Ann Clin Transl Neurol 2019 01 15;6(1):106-113. Epub 2018 Nov 15.

Department of Genetics and Metabolic Diseases Center for Clinical Genetics Hadassah Medical Center Jerusalem Israel.

Objective: To investigate the genetic basis of the recessive form of primary familial brain calcification and study pathways linking a novel gene with known dominant genes that cause the disease.

Methods: Whole exome sequencing and Sanger-based segregation analysis were used to identify possible disease causing mutations. Mutation pathogenicity was validated by structural protein modeling. Functional associations between the candidate gene, , and genes previously implicated in the disease were examined through phylogenetic profiling.

Results: We studied nine affected individuals from two unrelated families of Middle Eastern origin. The median age of symptom onset was 29.5 years (range 21-57 years) and dysarthria was the most common presenting symptom. We identified in the gene, a homozygous c.1233delC mutation in one family and c.1060_1062delGAC mutation in another. The first mutation results in protein truncation and the second in deletion of a highly conserved aspartic acid that is likely to disrupt binding of the protein with its substrate. Phylogenetic profiling analysis of the MYORG protein sequence suggests co-evolution with a number of calcium channels as well as other proteins related to regulation of anion transmembrane transport (False Discovery Rate, FDR < 10) and with PDCD6IP, a protein interacting with PDGFR which is known to be involved in the disease.

Interpretation: mutations are linked to a recessive form of primary familial brain calcification. This association was recently described in patients of Chinese ancestry. We suggest the possibility that mutations lead to calcification in a PDGFR -related pathway.
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http://dx.doi.org/10.1002/acn3.684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331209PMC
January 2019

Genotypes and phenotypes of patients with Lafora disease living in Germany.

Neurol Res Pract 2019 12;1. Epub 2019 Nov 12.

Center for Genomics and Transcriptomics (CeGaT) GmbH and Practice for Human Genetics, Tübingen, Germany.

Background: Lafora progressive myoclonus epilepsy (Lafora disease) is a rare, usually childhood-onset, fatal neurodegenerative disease caused by biallelic mutations in (Laforin) or (, Malin). The epidemiology of Lafora disease in Germany is largely unknown. The objective of this retrospective case series is to characterize the genotypes and phenotypes of patients with Lafora disease living in Germany.

Methods: The patients described in this case series initially had the suspected clinical diagnosis of Lafora disease, or unclassified progressive myoclonus epilepsy. Molecular genetic diagnostics including next generation sequencing-based diagnostic panel analysis or whole exome sequencing was performed.

Results: The parents of four out of the 11 patients are nonconsanguineous and of German origin while the other patients had consanguineous parents. Various variants were found in (six patients) and in (five patients). Eight variants have not been reported in the literature so far. The patients bearing novel variants had typical disease onset during adolescence and show classical disease courses.

Conclusions: This is the first larger case series of Lafora patients in Germany. Our data enable an approximation of the prevalence of manifest Lafora disease in Germany to 1,69 per 10 million people. Broader application of gene panel or whole-exome diagnostics helps clarifying unclassified progressive myoclonus epilepsy and establish an early diagnosis, which will be even more important as causal therapy approaches have been developed and are soon to be tested in a phase I study.
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http://dx.doi.org/10.1186/s42466-019-0040-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316188PMC
November 2019

deficiency in an intellectual disability, epilepsy, myoclonus, akathisia syndrome.

Ann Clin Transl Neurol 2018 Dec 24;5(12):1617-1621. Epub 2018 Oct 24.

Program in Genetics and Genome Biology The Hospital for Sick Children Toronto Ontario Canada.

We report a family of Saudi Arabian ancestry with two children presenting with global developmental delay, dystonia, disturbed sleep, and heat intolerance. By genome sequencing, we identified a nonsense variant in the first exon of that was homozygous in both affected individuals and was absent from, or heterozygous in, seven unaffected siblings. is highly expressed in the brain and a mouse model displays a neurological phenotype, implicating as a new disease gene for a neurological disorder.
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http://dx.doi.org/10.1002/acn3.677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6292187PMC
December 2018
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