- Email: [email protected]
Recent advances in epilepsy genetics
Accepted Manuscript Title: Recent advances in epilepsy genetics Authors: Alessandro Orsini, Federico Zara, Pasquale Striano PII: DOI: Reference:
S0304-3940(17)30402-0 http://dx.doi.org/doi:10.1016/j.neulet.2017.05.014 NSL 32821
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
20-12-2016 20-4-2017 8-5-2017
Please cite this article as: Alessandro Orsini, Federico Zara, Pasquale Striano, Recent advances in epilepsy genetics, Neuroscience Lettershttp://dx.doi.org/10.1016/j.neulet.2017.05.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
MINIREVIEW Neuroscience Letters Recent advances in epilepsy genetics 1
Alessandro Orsini, 2Federico Zara, 1Pasquale Striano
1
Pediatric Neurology and Muscular Diseases Unit, Department of Neurosciences, Rehabilitation,
Ophtalmology, Genetics, Maternal and Child Health, Institute "G. Gaslini" University of Genova, Genoa, Italy 2
Pediatric Neurology and Muscular Diseases Unit, Laboratory of Neurogenetics, Institute "G.
Gaslini", Genoa, Italy
The authors declare no conflicts of interest
CORRESPONDING AUTHOR: Alessandro Orsini, M.D, Pediatric Neurology and Neuromuscular Diseases Unit G. Gaslini Children’s Hospital Genova, Italy Phone: 01056362635 Fax: 010/8612070 [email protected] Highlights:
1: Several genetic tests are available for diagnostic purposes in epilepsy patients 2: Next-generation sequencing is revealing novel epilepsy genes 3: The identification of the genetic atiology may improve epilepsy treatment
ABSTRACT In last few years there has been rapid increase in the knowledge of epilepsy genetics. Nowadays, it is estimated that genetic epilepsies include over than 30 % of all epilepsy syndromes. Several genetic tests are now available for diagnostic purposes in clinical practice. In particular, next-generation sequencing has proven to be effective in revealing gene mutations causing epilepsies in up to a third of the patients. This has lead also to functional studies that have given insight into disease pathophysiology and consequently to
1
the identification of potential therapeutic targets opening the way of precision medicine for epilepsy patients. This minireview is focused on the most recent advances in genetics of epilepsies. We will also overview the modern genomic technologies and illustrate the diagnostic pathways in patients with genetic epilepsies. Finally, the potential implications for a personalized treatment (precision medicine) are also discussed. Key-words: next-generation sequencing; whole-exome sequencing; epilepsy; genetics, monogenic epilepsy; precision medicine 1. INTRODUCTION Epidemiological studies indicate that approximately 40–50 million people in the world suffer from epilepsy. Moreover, epilepsy is more frequent in infancy and elderly with an incidence of approximately 70 per 100 000 children aged younger than 2 years and it is estimated that the genetic epilepsies involve more than 0.4% of the general population, and constitute 30% of all epilepsies.1-3 Many different forms are defined as epileptic encephalopathies assuming that seizure activity plays a major role in developmental slowing or regression that follow seizure onset or exacerbation. 1,4 However, there is increasing evidence that seizures or interictal epileptiform activity contribute to exacerbate underlying brain dysfunction, beyond what expected from the underlying pathology alone. 4-6 In last few years, there has been rapid increase in the knowledge of epilepsy genetics.7 Most of these mutations involves the subunits of neuronal ion channels resulting in neuronal hyperexcitability or in reduction of inhibitory mechanisms, causing the recurrence of the seizures.8,9 However, several genes coding for different proteins have been associated recently with different types of epilepsy or epileptic encephalopathies.1,10 In particular, next-generation sequencing (NGS) techniques, i.e. whole exome sequencing (WES) and whole genome sequencing (WGS)-that have become considerably faster and more affordable over the past few years- are remarkably improving our understanding on the causes and pathophysiology, especially of epileptic encephalopathy.11-14
2
On the other hand, as these advanced technologies generate a huge amount of new data, it is crucial to provide clinicians with a comprehensive perspective that may favor their rationale use and insightful interpretation of the data into the clinical dimension, and improved capability of adopting personalized therapeutic strategies. Moreover, clinicians should be aware that each genetic technique has some limits and none of them is today able to scan the entire genome in all its complexity. This review is focused on the most recent advances in genetics of epilepsies. We will also illustrate the diagnostic pathways in patients with genetic epilepsies. Finally, the potential implications for a personalized treatment (precision medicine) are also discussed. 2. SEARCH STRATEGY AND SELECTION CRITERIA Data for this review were identified by searching through several databases (OMIM, HGMD, and EpilepsyGene) and publications on PubMed up to Dec 16 (keywords: epilepsy; epileptic syndrome; gene; genetics). Only papers published in English were reviewed. 3. MODERN GENETIC ANALYSIS TECHNIQUES 3.1. Array-CGH and other cytogenetic techniques
Although karyotype remains still an useful diagnostic test to identify chromosomal rearrangements, such as translocations or ring chromosomes (e.g., ring 14 and 20 chromosomes),15 array-comparative genomic hybridisation (array-CGH) is now the first-line genetic test in the investigation of ‘otherwise explained’ epilepsy, especially if the clinical picture includes dysmorphism, congenital anomalies, other neuropsychiatric features.16 Array-CGH allows to detect genomic microdeletions and microduplications that are invisible in a conventional karyotype.7,16,17 In addition, it is also able to define the exact genomic region altered and therefore the genes contained therein, improving genotype-phenotype correlations. However, the results of this analysis need to be carefully interpreted, due the uncertain clinical significance of copy number variants (CNVs) and its diagnostic impact is less evident when epilepsy is not associated with mental retardation.16,18,19 Altogether, rare CNVs, some of which involve known disease genes, contribute to at least 10% of childhood epilepsies and up to 5% of epileptic encephalopathies. 3
20
Moreover, the application of genome-wide microarrays in large cohorts of patients revealed that
large, recurrent deletions of chromosome 15q13.3, 15q11.2, 16p13.11 are presents in patients with focal or generalized epilepsy or epileptic encephalopathy more frequently than in the general population, representing a possible susceptibility factor.21-23 Fluorescence in situ hybridization (FISH) is reserved for the suspect of microdeletion syndromes/duplication and to better characterize the chromosomal abnormalities identified by other techniques (e.g. inv-dup 15). Finally, MLPA (multiplex ligation-dependent probe amplification) is used to identify intragenic deletions or duplications, such as sometimes observed in some patients with SCN1A or CDKL5 related-epilepsy who are negative on PCR sequencing or array-CGH. 24 3.2.
Next generation sequencing
PCR-Sanger sequencing is a rapid method for the determination of DNA sequences by means of primed synthesis with DNA polymerase. Over the last few years, it has been replaced by the next-generation sequencing (NGS) that allows the sequencing of several genes, quickly and cost effectively and has facilitated genetic research on epilepsy in the past few years. In fact, the NGS includes a variety of techniques that allow for the simultaneous sequencing of a large number of DNA segments, that can be the exons of a selected group of genes organized in panels (gene panels) or the entire exome.12,13,25 This technique is useful in the case of suspected single-gene defects with more than one gene involved in absence of multiorgan dysfunction or any dysmorphism. Whole-exome sequencing (WES) refers to the sequencing of part of the genome, namely exomes, which encodes proteins. Overall, the human exome includes about 20,000 genes which is associated with the majority of human disease. 11,26 WES analysis determines the profile of the identified gene variants and then a comparison with the polymorphic (non-pathogenic) variants distributed in the general population is performed to identify the possible pathogenic variants. The putative mutations are then be prioritized in relation to the occurrence of de novo (absent in the parents) and to the state of homozygosity (both gene copies suffering from the same mutation) or compound heterozygous (two different mutations in the same gene). NGS techniques are potentially very useful for the study of genetic epilepsies but, they generate a
4
variety of incidental findings, and the proper interpretation can be complicated (Table 1). However, the challenge of WGS lies within interpretation of detected variants and prediction of their pathogenicity. In addition, not all clinically relevant mutations (non-coding regulatory sequences, deletions/duplications of exons) are identified by this technique,12,25 e.g. mutations associated in noncoding parts of the genome, such as the dodecamer repeat upstream of the transcription start site of cystatin B, which is the cause of Unverricht–Lundborg type Progressive Myoclonus Epilepsy. 27 4. NOVEL FINDINGS WITH NGS APPROCH In the last few years, the wide application of NGS techniques has accelerated the identification of new genes responsible for rare monogenic epilepsies as well as of a number of epileptic encephalopathies in children.28,29 An exhaustive, recent update on epilepsy-associated genes according to their functions is available in Wang et al. 30 Most of the epileptogenic mutations have been so far identified ion channel genes, reinforcing the concept that several epilepsies are indeed channelopathies. 14,31 In particular, mutations in voltage-gated sodium channels genes are the most recognized cause of genetic epilepsy, 9,32 such as Dravet syndrome, the most deleterious epilepsy syndromes during childhood. This condition is commonly caused by de novo mutations in the SCN1A gene, encoding the alpha1-subunit of the neuronal voltage-gated sodium channel (SCN1A), which is the most clinically relevant among all the known epilepsy genes. Over 350 mutations have been so far identified with missense mutations being most common in GEFS+ and more deleterious mutations (nonsense, frameshift, deletions) representing the majority of the mutations. 33,34 In DS patients, the genetic change results in altered function of Nav1.1 sodium channels in neurons clustered throughout the brain.16,18 Seizure susceptibility in SCN1A mutation is caused by impaired (GABA)ergic firing in hippocampal interneurons and cerebellar Purkinje cells, which lowers the seizure threshold and the motor disorder presenting with ataxia.35 SCN2A encodes the major alpha subunit (Nav 1.2) of voltage- gated sodium channels. Mutations mainly lead to gain-of-function defects causing neuronal hyperexcitability as originally proposed by Scalmani et al 2006. 36 SCN2A mutations phenotype spectrum ranges from benign infantile neonatal seizures to a severe epileptic encephalopathy, often associated with other neurological features, such as movement disorder or ataxia and an increased risk of sudden
5
unexpected death in epilepsy (SUDEP). 37,38 Recently, mutations in SCN8A, encoding for the sodium channel alpha-subunit Nav 1.6, have been associated with epileptic encephalopathy and mental retardation. Subsequently, they have been reported also in milder epileptic phenotypes without intellectual disability, in other movement disorder conditions (paroxysmal dyskinesia, ataxia).39-41 The pathogenesis has been predominantly associated with gain-of-function mutations of SCN8A causing increased activation of the Nav 1.6 sodium channel.41 This large broad phenotype seems to be attributed to the expression of genes in different brain areas inducing dysfunction of neuronal network. 32 Among genes also the γ-aminobutyric acid (GABA) genes, that are implicated in the principal inhibitory mechanism in the central nervous system are associated with some idiopathic epilepsy syndromes, such as childhood absence epilepsy, autosomal dominant epilepsy with febrile seizures plus, and autosomal dominant Juvenile Myoclonic Epilepsy.42 Other channelopathies have also been recently identified in a number of patients with epilepsy syndromes ranging from benign neonatal familial seizures to severe epileptic encephalopathies include potassium voltage-gated channel subfamily (KCNQ) and hyperpolarization activated cyclic nucleotide gated channels (HCN). 43,44 Although mutations of ion channels are a main mechanism of genetic epilepsies, the use of highthroughput approaches to sequence DNA is revealing a landscape of mutations in genetic epilepsies, affecting a variety of genes involved in neuronal excitability, synaptic transmission, neuronal metabolism, or network development. These include mutations of protocadherin delta-2 subclass of the cadherin super family (PCDH19), syntaxin binding protein 1 (STXBP1), and the chromodomain helicase DNA binding protein 2 (CHD2) . 45,46 This increasing number of genes involved in a diversity of functional and developmental processes confirms that there is remarkable complexity underlying epileptogenesis. 5. IMPLICATIONS FOR MANAGEMENT Genetic counseling can be arranged once the genetic architecture and mode of inheritance of the epilepsy are fully understood (Figure 1). For example, the risk of recurrence in relatives of patients with PCDH19 mutations is quite different from Dravet syndrome patients carrying SCN1A mutations. Therefore, the
6
direct consequences for the individual and family of receiving a genetic diagnosis must be considered, even gene function and effects have not been yet clarify. 28,47 In addition, the discovery of a causative gene defect associated with a non-progressive course may reduce the need for further diagnostic investigations in the search for a progressive disorder at the biochemical and imaging level and allow more timely and straightforward treatment choices for specific conditions as well as avoiding needless investigations and inappropriate or unnecessary choices. There is, however, the need for a close collaboration between the geneticists and epileptologists to ensure the proper management of genetic investigations in patients with epilepsy. This interaction is crucial for both paediatric and adults patients towards the aim of an individualized (precision) medicine. Indeed, the advances in the genetics and neurobiology of the epilepsies are establishing the basis for a new era in the treatment, focused on each individual and their specific epilepsy, even later in life. 4.1 Precision medicine Precision medicine treatments represent a growing area of interest, focusing on reversing or circumventing the pathophysiological effects of specific gene mutations. Nevertheless, disease-specific treatments are currently available for only a minority of genetic epilepsies whereas for the large majority of patients, treatment options comprise the usual drug armamentarium that do not address the underlying biological mechanism. In addition, epileptologists well known that there is high variability in the response to antiepileptic treatment and that several factors may contribute to such variability. Variation in response to antiepileptic drug treatment may arise from genetic variation in a range of gene categories, including genes affecting drug pharmacokinetics, and drug pharmacodynamics, but also genes held to actually cause the epilepsy itself. Unfortunately, many findings are still controversial with anecdotal or less secure evidence and need further validation, e.g. variation in genes for transporter systems and antiepileptic drug targets.48 On the other hand, the increasing use of genetic sequencing could lead to a dramatic improvement of the effectiveness of epilepsy treatments, by targeting the biological mechanisms responsible for epilepsy in each specific individual. In fact, there are a few examples in which the discovery of the genetic defect
7
underlying a particular form of epilepsy may explain, in whole or in part, the response, whether positive or negative (paradoxical) to certain AEDs (Table 2).
Dravet syndrome, which is caused by mutations of the gene encoding the sodium channel (SCN1A) can be worsened by treatment with sodium-blocking drugs, such as carbamazepine and phenytoin, which should therefore be avoided. 2,49,50 Controversy is the use of lamotrigine (LTG) that works as a sodium channel and N-type calcium channel blocker. Lamotrigine has been reported to cause seizure exacerbation, which has led to avoidance of its use in patients with Dravet syndrome, but has been reported also a positive effect in some patients with Dravet Syndrome. This beneficial effect maybe explained with the action of LTG that involve hyperpolarization-activated cyclic nucleotide-gated channels. 51,52 Conversely, sodium blockers are considered first-choice therapy on epilepsy syndrome related to mutation of the gene SCN8A (another sodium channel gene) and KCNQ2. 2,53 Another example of precision medicine is the use of quinidine, an anti-arrhythmic drug in KCNT1-related epilepsies that in recent studies have been reported to be effective in seizure reduction or retigabine well known antiepileptic drugs most used in adult patients that selectively enhance the function of potassium channels formed by neuronal Kv7 subunits in KCNQ2-related epilepsies. 54 Although the discoveries in the field of genetic epilepsy syndrome have brought to the possibility of specific target management such as counseling, specific treatment that acts on the specific genetic defect and sometimes on the causative epilepsy’s gene mutation. 2,28 There are various associations between mutations of specific genes and rational treatment decisions. An example is the ketogenic diet in patients with Glut1-deficiency syndrome and mutations in SLC2A1. Glut1-deficiency syndrome is a genetic syndrome caused by insufficient transport of glucose from the blood to the brain which is characterized by the appearance of paroxysmal events in early childhood. The epileptic phenotype extends from mild cases with absence epilepsy to severe cases characterized by intractable epilepsy, infantile spasms with development delay. Classic phenotypes 8
include epileptic encephalopathy, developmental delay with complex movement disorders (dystonia, ataxia, and spasticity).55 The specific treatment is the ketogenic diet, an high-fat, lowcarbohydrate diet mimicking the fasting state. Nutritional fat is transformed into ketone bodies, which can be used as metabolic substrate for the brain when lacking glucose. On ketogenic diet significant seizure reduction was seen in children with the most severe epilepsies; seizure free or reduction was seen in 38% to 100% of children on the diet. 28,56 Moreover, early diagnosis and prompt treatment are important for prognosis. 56-58 Other examples are pyridoxal 5-phosphate dependant epilepsy due to mutation of the ALDH7A1 gene recently PNPO gene. 59-61 This enzyme converts α-aminoadipic semialdehyde (α-AASA) into α-aminoadipate (AAA), a critical step in the lysine metabolism of the brain and is involved in the metabolism of vitamin B6 of nutritional origin (in the form of pyridoxine and pyridoxamine) in active form, pyridoxal 5-phosphate (PLP). PLP is necessary in many processes including the production of neurotransmitters.60 This epilepsy syndrome is characterized by the onset of seizures shortly after birth, or in some cases, even intrafetal.62 AEDs which are usually given to control seizures, are ineffective in patients with PLPdependent epilepsy. These individuals respond instead to treatment with high dose on a daily use of PLP. If untreated can instead develop encephalopathy that can also have a fatal outcome.59,62 Another interesting finding concerns the association between genetic mutations in the gene DEPDC5, a gene involved in mTOR pathway and tuberous sclerosis, and various forms of epilepsy, tumor and neurodevelopmental disease both familial and sporadic with wide phenotypic spectrum.63 In these case a possibility is to use a specific drug known as rapamycin that act directly on mTOR pathway.64 In particular, tuberous sclerosis complex, a disease that brings to more than 80 % of drug resistant epilepsy patients other than disease in multiple organs, caused by mutations in TSC1 and TSC2 genes, encoding a complex with inhibitory activity against mTOR are susceptibility to rapamacin treatment. Early administration of the specific treatment can lead to better outcome not only in seizure but also in developmental delay. 65 5: CONCLUSION 9
In the last twenty years there have been enormous progress made with the development of molecular genetic techniques. Although ion channel genes represent the gene family most frequently causally related to epilepsy, other genes have gradually been associated with complex developmental epilepsy conditions, revealing the pathogenic role of mutations affecting diverse molecular pathways that regulate membrane excitability, synaptic plasticity, presynaptic neurotransmitter release, postsynaptic receptors, transporters, cell metabolism, and many formative steps in early brain development. Some of these discoveries are being followed by proof-of-concept laboratory studies that might open new pathways towards personalized treatment choices, not only for children but also for adult patients that may have a beneficial effect identifying the specific defects with a precision medicine treatment and a specific counselling for procreation. In addition, this increasing knowledge has led to the design of new drugs targeted to specific pathogenic mechanisms, or to a specific action of mutated proteins, up to a gene replacement therapy. However, further research will likely require an interdisciplinary and international collaboration approach, combining basic research with clinical studies. References
1. McTague A, Howell KB, Cross JH, Kurian MA, Scheffer IE. The genetic landscape of the epileptic encephalopathies of infancy and childhood. The Lancet Neurology 2016; 15(3): 304-16. 2. Striano P, Vari MS, Mazzocchetti C, Verrotti A, Zara F. Management of genetic epilepsies: From empirical treatment to precision medicine. Pharmacological research 2016; 107: 426-9. 3. International League Against Epilepsy Consortium on Complex Epilepsies. Electronic address e-auea. Genetic determinants of common epilepsies: a meta-analysis of genome-wide association studies. The Lancet Neurology 2014; 13(9): 893-903. 4. Jehi L, Wyllie E, Devinsky O. Epileptic encephalopathies: Optimizing seizure control and developmental outcome. Epilepsia 2015; 56(10): 1486-9. 5. Shao LR, Stafstrom CE. Pediatric Epileptic Encephalopathies: Pathophysiology and Animal Models. Seminars in pediatric neurology 2016; 23(2): 98-107. 6. Auvin S, Cilio MR, Vezzani A. Current understanding and neurobiology of epileptic encephalopathies. Neurobiology of disease 2016; 92(Pt A): 72-89. 7. Sisodiya SM. Genetic screening and diagnosis in epilepsy? Current opinion in neurology 2015; 28(2): 136-42. 8. Villa C, Combi R. Potassium Channels and Human Epileptic Phenotypes: An Updated Overview. Frontiers in cellular neuroscience 2016; 10: 81. 9. Kaplan DI, Isom LL, Petrou S. Role of Sodium Channels in Epilepsy. Cold Spring Harbor perspectives in medicine 2016; 6(6). 10. Mantegazza M, Rusconi R, Scalmani P, Avanzini G, Franceschetti S. Epileptogenic ion channel mutations: from bedside to bench and, hopefully, back again. Epilepsy research 2010; 92(1): 1-29. 10
11. Warman Chardon J, Beaulieu C, Hartley T, Boycott KM, Dyment DA. Axons to Exons: the Molecular Diagnosis of Rare Neurological Diseases by Next-Generation Sequencing. Current neurology and neuroscience reports 2015; 15(9): 64. 12. Moller RS, Dahl HA, Helbig I. The contribution of next generation sequencing to epilepsy genetics. Expert review of molecular diagnostics 2015; 15(12): 1531-8. 13. Guerreiro R, Bras J, Hardy J, Singleton A. Next generation sequencing techniques in neurological diseases: redefining clinical and molecular associations. Human molecular genetics 2014; 23(R1): R47-53. 14. Jiang T, Tan MS, Tan L, Yu JT. Application of next-generation sequencing technologies in Neurology. Annals of translational medicine 2014; 2(12): 125. 15. Ream MA, Mikati MA. Clinical utility of genetic testing in pediatric drug-resistant epilepsy: a pilot study. Epilepsy & behavior : E&B 2014; 37: 241-8. 16. Kharbanda M, Tolmie J, Joss S. How to use... microarray comparative genomic hybridisation to investigate developmental disorders. Archives of disease in childhood Education and practice edition 2015; 100(1): 24-9. 17. Spreiz A, Haberlandt E, Baumann M, et al. Chromosomal microaberrations in patients with epilepsy, intellectual disability, and congenital anomalies. Clinical genetics 2014; 86(4): 361-6. 18. Olson H, Shen Y, Avallone J, et al. Copy number variation plays an important role in clinical epilepsy. Annals of neurology 2014; 75(6): 943-58. 19. Striano P, Coppola A, Paravidino R, et al. Clinical significance of rare copy number variations in epilepsy: a case-control survey using microarray-based comparative genomic hybridization. Archives of neurology 2012; 69(3): 322-30. 20. Copy number variant analysis from exome data in 349 patients with epileptic encephalopathy. Annals of neurology 2015; 78(2): 323-8. 21. Bonuccelli A, Valetto A, Orsini A, et al. Maternally derived 15q11.2-q13.1 duplication in a child with Lennox-Gastaut-type epilepsy and dysmorphic features: Clinical-genetic characterization of the family and review of the literature. American journal of medical genetics Part A 2016. 22. Mefford HC, Muhle H, Ostertag P, et al. Genome-wide copy number variation in epilepsy: novel susceptibility loci in idiopathic generalized and focal epilepsies. PLoS genetics 2010; 6(5): e1000962. 23. de Kovel CG, Trucks H, Helbig I, et al. Recurrent microdeletions at 15q11.2 and 16p13.11 predispose to idiopathic generalized epilepsies. Brain : a journal of neurology 2010; 133(Pt 1): 2332. 24. Stuppia L, Antonucci I, Palka G, Gatta V. Use of the MLPA assay in the molecular diagnosis of gene copy number alterations in human genetic diseases. International journal of molecular sciences 2012; 13(3): 3245-76. 25. Myers CT, Mefford HC. Advancing epilepsy genetics in the genomic era. Genome medicine 2015; 7: 91. 26. Kadalayil L, Rafiq S, Rose-Zerilli MJ, et al. Exome sequence read depth methods for identifying copy number changes. Briefings in bioinformatics 2015; 16(3): 380-92. 27. Minassian BA, Striano P, Avanzini G. Progressive Myoclonus Epilepsy: The GeneEmpowered Era. Epileptic disorders : international epilepsy journal with videotape 2016; 18(S2): 1-2. 28. Thomas RH, Berkovic SF. The hidden genetics of epilepsy-a clinically important new paradigm. Nature reviews Neurology 2014; 10(5): 283-92. 29. Parrini E, Marini C, Mei D, et al. Diagnostic Targeted Resequencing in 349 Patients with Drug-Resistant Pediatric Epilepsies Identifies Causative Mutations in 30 Different Genes. Human mutation 2016. 30. Wang J, Lin ZJ, Liu L, et al. Epilepsy-associated genes. Seizure 2017; 44: 11-20.
11
31. Mantegazza M RR, Cestèle S. Mutations of Ion Channels in Genetic Epilepsies. Epilepsy Towards the Next Decade - New Trends and Hopes in Epileptology - Springer Science+Business Media 2015: 15-34. 32. de Lera Ruiz M, Kraus RL. Voltage-Gated Sodium Channels: Structure, Function, Pharmacology, and Clinical Indications. Journal of medicinal chemistry 2015; 58(18): 7093-118. 33. Camfield P, Camfield C. Febrile seizures and genetic epilepsy with febrile seizures plus (GEFS+). Epileptic disorders : international epilepsy journal with videotape 2015; 17(2): 124-33. 34. Djemie T, Weckhuysen S, von Spiczak S, et al. Pitfalls in genetic testing: the story of missed SCN1A mutations. Molecular genetics & genomic medicine 2016; 4(4): 457-64. 35. Brunklaus A, Zuberi SM. Dravet syndrome--from epileptic encephalopathy to channelopathy. Epilepsia 2014; 55(7): 979-84. 36. Scalmani P, Rusconi R, Armatura E, et al. Effects in neocortical neurons of mutations of the Na(v)1.2 Na+ channel causing benign familial neonatal-infantile seizures. The Journal of neuroscience : the official journal of the Society for Neuroscience 2006; 26(40): 10100-9. 37. Syrbe S, Zhorov BS, Bertsche A, et al. Phenotypic Variability from Benign Infantile Epilepsy to Ohtahara Syndrome Associated with a Novel Mutation in SCN2A. Molecular syndromology 2016; 7(4): 182-8. 38. Schwarz N, Hahn A, Bast T, et al. Mutations in the sodium channel gene SCN2A cause neonatal epilepsy with late-onset episodic ataxia. Journal of neurology 2016; 263(2): 334-43. 39. Gardella E, Becker F, Moller RS, et al. Benign infantile seizures and paroxysmal dyskinesia caused by an SCN8A mutation. Annals of neurology 2016; 79(3): 428-36. 40. Anand G, Collett-White F, Orsini A, et al. Autosomal dominant SCN8A mutation with an unusually mild phenotype. European journal of paediatric neurology : EJPN : official journal of the European Paediatric Neurology Society; 20(5): 761-5. 41. Meisler MH, Helman G, Hammer MF, et al. SCN8A encephalopathy: Research progress and prospects. Epilepsia 2016; 57(7): 1027-35. 42. Galanopoulou AS. Mutations affecting GABAergic signaling in seizures and epilepsy. Pflugers Arch 2010; 460(2): 505-23. 43. Nava C, Dalle C, Rastetter A, et al. De novo mutations in HCN1 cause early infantile epileptic encephalopathy. Nature genetics 2014; 46(6): 640-5. 44. Allen NM, Mannion M, Conroy J, et al. The variable phenotypes of KCNQ-related epilepsy. Epilepsia 2014; 55(9): e99-105. 45. Thomas RH, Zhang LM, Carvill GL, et al. CHD2 myoclonic encephalopathy is frequently associated with self-induced seizures. Neurology 2015; 84(9): 951-8. 46. Galizia EC, Myers CT, Leu C, et al. CHD2 variants are a risk factor for photosensitivity in epilepsy. Brain : a journal of neurology 2015; 138(Pt 5): 1198-207. 47. Trivisano M, Pietrafusa N, Ciommo V, et al. PCDH19-related epilepsy and Dravet Syndrome: Face-off between two early-onset epilepsies with fever sensitivity. Epilepsy research 2016; 125: 32-6. 48. Balestrini S, Sisodiya SM. Pharmacogenomics in epilepsy. Neuroscience letters 2017. 49. Wallace A, Wirrell E, Kenney-Jung DL. Pharmacotherapy for Dravet Syndrome. Paediatric drugs 2016; 18(3): 197-208. 50. Wirrell EC. Treatment of Dravet Syndrome. The Canadian journal of neurological sciences Le journal canadien des sciences neurologiques 2016; 43 Suppl 3: S13-8. 51. Dalic L, Mullen SA, Roulet Perez E, Scheffer I. Lamotrigine can be beneficial in patients with Dravet syndrome. Developmental medicine and child neurology 2015; 57(2): 200-2. 52. Andrade DM. Dravet syndrome, lamotrigine, and personalized medicine. Developmental medicine and child neurology 2015; 57(2): 118-9. 53. Boerma RS, Braun KP, van den Broek MP, et al. Remarkable Phenytoin Sensitivity in 4 Children with SCN8A-related Epilepsy: A Molecular Neuropharmacological Approach. Neurotherapeutics 2016; 13(1): 192-7. 12
54. Vezyroglou K, Cross JH. Targeted Treatment in Childhood Epilepsy Syndromes. Current treatment options in neurology 2016; 18(6): 29. 55. Lee HH, Hur YJ. Glucose transport 1 deficiency presenting as infantile spasms with a mutation identified in exon 9 of SLC2A1. Korean journal of pediatrics 2016; 59(Suppl 1): S29S31. 56. Ramm-Pettersen A, Nakken KO, Haavardsholm KC, Selmer KK. Occurrence of GLUT1 deficiency syndrome in patients treated with ketogenic diet. Epilepsy & behavior : E&B 2014; 32: 76-8. 57. Gumus H, Bayram AK, Kardas F, et al. The Effects of Ketogenic Diet on Seizures, Cognitive Functions, and Other Neurological Disorders in Classical Phenotype of Glucose Transporter 1 Deficiency Syndrome. Neuropediatrics 2015; 46(5): 313-20. 58. Akman CI, Yu J, Alter A, Engelstad K, De Vivo DC. Diagnosing Glucose Transporter 1 Deficiency at Initial Presentation Facilitates Early Treatment. The Journal of pediatrics 2016; 171: 220-6. 59. van Karnebeek CD, Tiebout SA, Niermeijer J, et al. Pyridoxine-Dependent Epilepsy: An Expanding Clinical Spectrum. Pediatric neurology 2016; 59: 6-12. 60. Coci EG, Codutti L, Fink C, et al. Novel homozygous missense mutation in ALDH7A1 causes neonatal pyridoxine dependent epilepsy. Molecular and cellular probes 2016. 61. Baumgart A, Spiczak S, Verhoeven-Duif NM, et al. Atypical vitamin B6 deficiency: a rare cause of unexplained neonatal and infantile epilepsies. Journal of child neurology 2014; 29(5): 7047. 62. Jaeger B, Abeling NG, Salomons GS, et al. Pyridoxine responsive epilepsy caused by a novel homozygous PNPO mutation. Molecular genetics and metabolism reports 2016; 6: 60-3. 63. Dibbens LM, de Vries B, Donatello S, et al. Mutations in DEPDC5 cause familial focal epilepsy with variable foci. Nature genetics 2013; 45(5): 546-51. 64. Sadowski K, Kotulska-Jozwiak K, Jozwiak S. Role of mTOR inhibitors in epilepsy treatment. Pharmacol Rep 2015; 67(3): 636-46. 65. Jeong A, Wong M. mTOR Inhibitors in Children: Current Indications and Future Directions in Neurology. Current neurology and neuroscience reports 2016; 16(12): 102. Figure Caption
13
Figure 1: Example of diagnostic algorithm for genetic epilepsies.
14
Table 1. Diagnostic tests for epilepsy patients. Diagnostic test
Description
When to use it
Identify single nucleotide polymorphisms (SNP arrays) or to determine chromosomal
Epilepsy with developmental delay,
rearrangements submicroscopic (array-CGH) as
dysmorphism, ASD
Array-CGH
CNVs.
Single gene
Detects changes in the gene and if it causes amino
sequencing
acid alterations
Suspected single-gene defect (e.g. SLC2A1 in Glut-1 deficiency)
Duplication Suspicious of a single gene defect deletion of a
CNV of a single gene when sequencing is inconclusive
single gene Research of a
On parents to understand if an Sequencing of a specific mutation
specific mutation
unknown mutation is pathological
Targeted-
Sequencing and duplication/deletion research of a
resequencing
gene panel for a specific disease
disease with more genes involved
Fluorescent in situ Probes that analyse specific chromosome’s hybridization
Confirmation of a duplication/deletion portions
(FISH) Analysis of all chromosome for big
Patients with dysmorphism and/or
duplication/deletion
multiorgan dysfunction
Sequencing of all DNA only for codifying regions
Suspected genetic aetiology with
(exons) or all regions (genome)
otherwise normal investigations
Karyotype
Whole-exome and genome sequencing
15
Table 2. Examples of precision medicine for genetic epilepsies. Gene
Epilepsy syndrome
Possible treatment(s)
SCN1A
Dravet syndrome
Avoid sodium channel blockers
Early-onset epileptic
Sodium channel blockers
encephalopathy
(carbamazepine, phenytoin)
SCN8A
KCNQ2
Early-onset epileptic encephalopathy
Potassium channel openers (retigabine) or sodium channel blockers (carbamazepine) Potassium channel openers
KCNT1
Migrating partial epilepsy of
(quinidine)
infancy
NMDA (N-methyl-D-aspartate) GRIN2A
SCL2A1 TSC1/TSC2 ALDH7A1
Early-onset epileptic
receptor antagonists
encephalopathy
(memantine)
GLUT-1 deficiency
Ketogenic Diet
Tuberous sclerosis complex Pyridoxine dependency
16
Rapamicin and analogs B6 vitamin
S0304-3940(17)30402-0 http://dx.doi.org/doi:10.1016/j.neulet.2017.05.014 NSL 32821
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
20-12-2016 20-4-2017 8-5-2017
Please cite this article as: Alessandro Orsini, Federico Zara, Pasquale Striano, Recent advances in epilepsy genetics, Neuroscience Lettershttp://dx.doi.org/10.1016/j.neulet.2017.05.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
MINIREVIEW Neuroscience Letters Recent advances in epilepsy genetics 1
Alessandro Orsini, 2Federico Zara, 1Pasquale Striano
1
Pediatric Neurology and Muscular Diseases Unit, Department of Neurosciences, Rehabilitation,
Ophtalmology, Genetics, Maternal and Child Health, Institute "G. Gaslini" University of Genova, Genoa, Italy 2
Pediatric Neurology and Muscular Diseases Unit, Laboratory of Neurogenetics, Institute "G.
Gaslini", Genoa, Italy
The authors declare no conflicts of interest
CORRESPONDING AUTHOR: Alessandro Orsini, M.D, Pediatric Neurology and Neuromuscular Diseases Unit G. Gaslini Children’s Hospital Genova, Italy Phone: 01056362635 Fax: 010/8612070 [email protected] Highlights:
1: Several genetic tests are available for diagnostic purposes in epilepsy patients 2: Next-generation sequencing is revealing novel epilepsy genes 3: The identification of the genetic atiology may improve epilepsy treatment
ABSTRACT In last few years there has been rapid increase in the knowledge of epilepsy genetics. Nowadays, it is estimated that genetic epilepsies include over than 30 % of all epilepsy syndromes. Several genetic tests are now available for diagnostic purposes in clinical practice. In particular, next-generation sequencing has proven to be effective in revealing gene mutations causing epilepsies in up to a third of the patients. This has lead also to functional studies that have given insight into disease pathophysiology and consequently to
1
the identification of potential therapeutic targets opening the way of precision medicine for epilepsy patients. This minireview is focused on the most recent advances in genetics of epilepsies. We will also overview the modern genomic technologies and illustrate the diagnostic pathways in patients with genetic epilepsies. Finally, the potential implications for a personalized treatment (precision medicine) are also discussed. Key-words: next-generation sequencing; whole-exome sequencing; epilepsy; genetics, monogenic epilepsy; precision medicine 1. INTRODUCTION Epidemiological studies indicate that approximately 40–50 million people in the world suffer from epilepsy. Moreover, epilepsy is more frequent in infancy and elderly with an incidence of approximately 70 per 100 000 children aged younger than 2 years and it is estimated that the genetic epilepsies involve more than 0.4% of the general population, and constitute 30% of all epilepsies.1-3 Many different forms are defined as epileptic encephalopathies assuming that seizure activity plays a major role in developmental slowing or regression that follow seizure onset or exacerbation. 1,4 However, there is increasing evidence that seizures or interictal epileptiform activity contribute to exacerbate underlying brain dysfunction, beyond what expected from the underlying pathology alone. 4-6 In last few years, there has been rapid increase in the knowledge of epilepsy genetics.7 Most of these mutations involves the subunits of neuronal ion channels resulting in neuronal hyperexcitability or in reduction of inhibitory mechanisms, causing the recurrence of the seizures.8,9 However, several genes coding for different proteins have been associated recently with different types of epilepsy or epileptic encephalopathies.1,10 In particular, next-generation sequencing (NGS) techniques, i.e. whole exome sequencing (WES) and whole genome sequencing (WGS)-that have become considerably faster and more affordable over the past few years- are remarkably improving our understanding on the causes and pathophysiology, especially of epileptic encephalopathy.11-14
2
On the other hand, as these advanced technologies generate a huge amount of new data, it is crucial to provide clinicians with a comprehensive perspective that may favor their rationale use and insightful interpretation of the data into the clinical dimension, and improved capability of adopting personalized therapeutic strategies. Moreover, clinicians should be aware that each genetic technique has some limits and none of them is today able to scan the entire genome in all its complexity. This review is focused on the most recent advances in genetics of epilepsies. We will also illustrate the diagnostic pathways in patients with genetic epilepsies. Finally, the potential implications for a personalized treatment (precision medicine) are also discussed. 2. SEARCH STRATEGY AND SELECTION CRITERIA Data for this review were identified by searching through several databases (OMIM, HGMD, and EpilepsyGene) and publications on PubMed up to Dec 16 (keywords: epilepsy; epileptic syndrome; gene; genetics). Only papers published in English were reviewed. 3. MODERN GENETIC ANALYSIS TECHNIQUES 3.1. Array-CGH and other cytogenetic techniques
Although karyotype remains still an useful diagnostic test to identify chromosomal rearrangements, such as translocations or ring chromosomes (e.g., ring 14 and 20 chromosomes),15 array-comparative genomic hybridisation (array-CGH) is now the first-line genetic test in the investigation of ‘otherwise explained’ epilepsy, especially if the clinical picture includes dysmorphism, congenital anomalies, other neuropsychiatric features.16 Array-CGH allows to detect genomic microdeletions and microduplications that are invisible in a conventional karyotype.7,16,17 In addition, it is also able to define the exact genomic region altered and therefore the genes contained therein, improving genotype-phenotype correlations. However, the results of this analysis need to be carefully interpreted, due the uncertain clinical significance of copy number variants (CNVs) and its diagnostic impact is less evident when epilepsy is not associated with mental retardation.16,18,19 Altogether, rare CNVs, some of which involve known disease genes, contribute to at least 10% of childhood epilepsies and up to 5% of epileptic encephalopathies. 3
20
Moreover, the application of genome-wide microarrays in large cohorts of patients revealed that
large, recurrent deletions of chromosome 15q13.3, 15q11.2, 16p13.11 are presents in patients with focal or generalized epilepsy or epileptic encephalopathy more frequently than in the general population, representing a possible susceptibility factor.21-23 Fluorescence in situ hybridization (FISH) is reserved for the suspect of microdeletion syndromes/duplication and to better characterize the chromosomal abnormalities identified by other techniques (e.g. inv-dup 15). Finally, MLPA (multiplex ligation-dependent probe amplification) is used to identify intragenic deletions or duplications, such as sometimes observed in some patients with SCN1A or CDKL5 related-epilepsy who are negative on PCR sequencing or array-CGH. 24 3.2.
Next generation sequencing
PCR-Sanger sequencing is a rapid method for the determination of DNA sequences by means of primed synthesis with DNA polymerase. Over the last few years, it has been replaced by the next-generation sequencing (NGS) that allows the sequencing of several genes, quickly and cost effectively and has facilitated genetic research on epilepsy in the past few years. In fact, the NGS includes a variety of techniques that allow for the simultaneous sequencing of a large number of DNA segments, that can be the exons of a selected group of genes organized in panels (gene panels) or the entire exome.12,13,25 This technique is useful in the case of suspected single-gene defects with more than one gene involved in absence of multiorgan dysfunction or any dysmorphism. Whole-exome sequencing (WES) refers to the sequencing of part of the genome, namely exomes, which encodes proteins. Overall, the human exome includes about 20,000 genes which is associated with the majority of human disease. 11,26 WES analysis determines the profile of the identified gene variants and then a comparison with the polymorphic (non-pathogenic) variants distributed in the general population is performed to identify the possible pathogenic variants. The putative mutations are then be prioritized in relation to the occurrence of de novo (absent in the parents) and to the state of homozygosity (both gene copies suffering from the same mutation) or compound heterozygous (two different mutations in the same gene). NGS techniques are potentially very useful for the study of genetic epilepsies but, they generate a
4
variety of incidental findings, and the proper interpretation can be complicated (Table 1). However, the challenge of WGS lies within interpretation of detected variants and prediction of their pathogenicity. In addition, not all clinically relevant mutations (non-coding regulatory sequences, deletions/duplications of exons) are identified by this technique,12,25 e.g. mutations associated in noncoding parts of the genome, such as the dodecamer repeat upstream of the transcription start site of cystatin B, which is the cause of Unverricht–Lundborg type Progressive Myoclonus Epilepsy. 27 4. NOVEL FINDINGS WITH NGS APPROCH In the last few years, the wide application of NGS techniques has accelerated the identification of new genes responsible for rare monogenic epilepsies as well as of a number of epileptic encephalopathies in children.28,29 An exhaustive, recent update on epilepsy-associated genes according to their functions is available in Wang et al. 30 Most of the epileptogenic mutations have been so far identified ion channel genes, reinforcing the concept that several epilepsies are indeed channelopathies. 14,31 In particular, mutations in voltage-gated sodium channels genes are the most recognized cause of genetic epilepsy, 9,32 such as Dravet syndrome, the most deleterious epilepsy syndromes during childhood. This condition is commonly caused by de novo mutations in the SCN1A gene, encoding the alpha1-subunit of the neuronal voltage-gated sodium channel (SCN1A), which is the most clinically relevant among all the known epilepsy genes. Over 350 mutations have been so far identified with missense mutations being most common in GEFS+ and more deleterious mutations (nonsense, frameshift, deletions) representing the majority of the mutations. 33,34 In DS patients, the genetic change results in altered function of Nav1.1 sodium channels in neurons clustered throughout the brain.16,18 Seizure susceptibility in SCN1A mutation is caused by impaired (GABA)ergic firing in hippocampal interneurons and cerebellar Purkinje cells, which lowers the seizure threshold and the motor disorder presenting with ataxia.35 SCN2A encodes the major alpha subunit (Nav 1.2) of voltage- gated sodium channels. Mutations mainly lead to gain-of-function defects causing neuronal hyperexcitability as originally proposed by Scalmani et al 2006. 36 SCN2A mutations phenotype spectrum ranges from benign infantile neonatal seizures to a severe epileptic encephalopathy, often associated with other neurological features, such as movement disorder or ataxia and an increased risk of sudden
5
unexpected death in epilepsy (SUDEP). 37,38 Recently, mutations in SCN8A, encoding for the sodium channel alpha-subunit Nav 1.6, have been associated with epileptic encephalopathy and mental retardation. Subsequently, they have been reported also in milder epileptic phenotypes without intellectual disability, in other movement disorder conditions (paroxysmal dyskinesia, ataxia).39-41 The pathogenesis has been predominantly associated with gain-of-function mutations of SCN8A causing increased activation of the Nav 1.6 sodium channel.41 This large broad phenotype seems to be attributed to the expression of genes in different brain areas inducing dysfunction of neuronal network. 32 Among genes also the γ-aminobutyric acid (GABA) genes, that are implicated in the principal inhibitory mechanism in the central nervous system are associated with some idiopathic epilepsy syndromes, such as childhood absence epilepsy, autosomal dominant epilepsy with febrile seizures plus, and autosomal dominant Juvenile Myoclonic Epilepsy.42 Other channelopathies have also been recently identified in a number of patients with epilepsy syndromes ranging from benign neonatal familial seizures to severe epileptic encephalopathies include potassium voltage-gated channel subfamily (KCNQ) and hyperpolarization activated cyclic nucleotide gated channels (HCN). 43,44 Although mutations of ion channels are a main mechanism of genetic epilepsies, the use of highthroughput approaches to sequence DNA is revealing a landscape of mutations in genetic epilepsies, affecting a variety of genes involved in neuronal excitability, synaptic transmission, neuronal metabolism, or network development. These include mutations of protocadherin delta-2 subclass of the cadherin super family (PCDH19), syntaxin binding protein 1 (STXBP1), and the chromodomain helicase DNA binding protein 2 (CHD2) . 45,46 This increasing number of genes involved in a diversity of functional and developmental processes confirms that there is remarkable complexity underlying epileptogenesis. 5. IMPLICATIONS FOR MANAGEMENT Genetic counseling can be arranged once the genetic architecture and mode of inheritance of the epilepsy are fully understood (Figure 1). For example, the risk of recurrence in relatives of patients with PCDH19 mutations is quite different from Dravet syndrome patients carrying SCN1A mutations. Therefore, the
6
direct consequences for the individual and family of receiving a genetic diagnosis must be considered, even gene function and effects have not been yet clarify. 28,47 In addition, the discovery of a causative gene defect associated with a non-progressive course may reduce the need for further diagnostic investigations in the search for a progressive disorder at the biochemical and imaging level and allow more timely and straightforward treatment choices for specific conditions as well as avoiding needless investigations and inappropriate or unnecessary choices. There is, however, the need for a close collaboration between the geneticists and epileptologists to ensure the proper management of genetic investigations in patients with epilepsy. This interaction is crucial for both paediatric and adults patients towards the aim of an individualized (precision) medicine. Indeed, the advances in the genetics and neurobiology of the epilepsies are establishing the basis for a new era in the treatment, focused on each individual and their specific epilepsy, even later in life. 4.1 Precision medicine Precision medicine treatments represent a growing area of interest, focusing on reversing or circumventing the pathophysiological effects of specific gene mutations. Nevertheless, disease-specific treatments are currently available for only a minority of genetic epilepsies whereas for the large majority of patients, treatment options comprise the usual drug armamentarium that do not address the underlying biological mechanism. In addition, epileptologists well known that there is high variability in the response to antiepileptic treatment and that several factors may contribute to such variability. Variation in response to antiepileptic drug treatment may arise from genetic variation in a range of gene categories, including genes affecting drug pharmacokinetics, and drug pharmacodynamics, but also genes held to actually cause the epilepsy itself. Unfortunately, many findings are still controversial with anecdotal or less secure evidence and need further validation, e.g. variation in genes for transporter systems and antiepileptic drug targets.48 On the other hand, the increasing use of genetic sequencing could lead to a dramatic improvement of the effectiveness of epilepsy treatments, by targeting the biological mechanisms responsible for epilepsy in each specific individual. In fact, there are a few examples in which the discovery of the genetic defect
7
underlying a particular form of epilepsy may explain, in whole or in part, the response, whether positive or negative (paradoxical) to certain AEDs (Table 2).
Dravet syndrome, which is caused by mutations of the gene encoding the sodium channel (SCN1A) can be worsened by treatment with sodium-blocking drugs, such as carbamazepine and phenytoin, which should therefore be avoided. 2,49,50 Controversy is the use of lamotrigine (LTG) that works as a sodium channel and N-type calcium channel blocker. Lamotrigine has been reported to cause seizure exacerbation, which has led to avoidance of its use in patients with Dravet syndrome, but has been reported also a positive effect in some patients with Dravet Syndrome. This beneficial effect maybe explained with the action of LTG that involve hyperpolarization-activated cyclic nucleotide-gated channels. 51,52 Conversely, sodium blockers are considered first-choice therapy on epilepsy syndrome related to mutation of the gene SCN8A (another sodium channel gene) and KCNQ2. 2,53 Another example of precision medicine is the use of quinidine, an anti-arrhythmic drug in KCNT1-related epilepsies that in recent studies have been reported to be effective in seizure reduction or retigabine well known antiepileptic drugs most used in adult patients that selectively enhance the function of potassium channels formed by neuronal Kv7 subunits in KCNQ2-related epilepsies. 54 Although the discoveries in the field of genetic epilepsy syndrome have brought to the possibility of specific target management such as counseling, specific treatment that acts on the specific genetic defect and sometimes on the causative epilepsy’s gene mutation. 2,28 There are various associations between mutations of specific genes and rational treatment decisions. An example is the ketogenic diet in patients with Glut1-deficiency syndrome and mutations in SLC2A1. Glut1-deficiency syndrome is a genetic syndrome caused by insufficient transport of glucose from the blood to the brain which is characterized by the appearance of paroxysmal events in early childhood. The epileptic phenotype extends from mild cases with absence epilepsy to severe cases characterized by intractable epilepsy, infantile spasms with development delay. Classic phenotypes 8
include epileptic encephalopathy, developmental delay with complex movement disorders (dystonia, ataxia, and spasticity).55 The specific treatment is the ketogenic diet, an high-fat, lowcarbohydrate diet mimicking the fasting state. Nutritional fat is transformed into ketone bodies, which can be used as metabolic substrate for the brain when lacking glucose. On ketogenic diet significant seizure reduction was seen in children with the most severe epilepsies; seizure free or reduction was seen in 38% to 100% of children on the diet. 28,56 Moreover, early diagnosis and prompt treatment are important for prognosis. 56-58 Other examples are pyridoxal 5-phosphate dependant epilepsy due to mutation of the ALDH7A1 gene recently PNPO gene. 59-61 This enzyme converts α-aminoadipic semialdehyde (α-AASA) into α-aminoadipate (AAA), a critical step in the lysine metabolism of the brain and is involved in the metabolism of vitamin B6 of nutritional origin (in the form of pyridoxine and pyridoxamine) in active form, pyridoxal 5-phosphate (PLP). PLP is necessary in many processes including the production of neurotransmitters.60 This epilepsy syndrome is characterized by the onset of seizures shortly after birth, or in some cases, even intrafetal.62 AEDs which are usually given to control seizures, are ineffective in patients with PLPdependent epilepsy. These individuals respond instead to treatment with high dose on a daily use of PLP. If untreated can instead develop encephalopathy that can also have a fatal outcome.59,62 Another interesting finding concerns the association between genetic mutations in the gene DEPDC5, a gene involved in mTOR pathway and tuberous sclerosis, and various forms of epilepsy, tumor and neurodevelopmental disease both familial and sporadic with wide phenotypic spectrum.63 In these case a possibility is to use a specific drug known as rapamycin that act directly on mTOR pathway.64 In particular, tuberous sclerosis complex, a disease that brings to more than 80 % of drug resistant epilepsy patients other than disease in multiple organs, caused by mutations in TSC1 and TSC2 genes, encoding a complex with inhibitory activity against mTOR are susceptibility to rapamacin treatment. Early administration of the specific treatment can lead to better outcome not only in seizure but also in developmental delay. 65 5: CONCLUSION 9
In the last twenty years there have been enormous progress made with the development of molecular genetic techniques. Although ion channel genes represent the gene family most frequently causally related to epilepsy, other genes have gradually been associated with complex developmental epilepsy conditions, revealing the pathogenic role of mutations affecting diverse molecular pathways that regulate membrane excitability, synaptic plasticity, presynaptic neurotransmitter release, postsynaptic receptors, transporters, cell metabolism, and many formative steps in early brain development. Some of these discoveries are being followed by proof-of-concept laboratory studies that might open new pathways towards personalized treatment choices, not only for children but also for adult patients that may have a beneficial effect identifying the specific defects with a precision medicine treatment and a specific counselling for procreation. In addition, this increasing knowledge has led to the design of new drugs targeted to specific pathogenic mechanisms, or to a specific action of mutated proteins, up to a gene replacement therapy. However, further research will likely require an interdisciplinary and international collaboration approach, combining basic research with clinical studies. References
1. McTague A, Howell KB, Cross JH, Kurian MA, Scheffer IE. The genetic landscape of the epileptic encephalopathies of infancy and childhood. The Lancet Neurology 2016; 15(3): 304-16. 2. Striano P, Vari MS, Mazzocchetti C, Verrotti A, Zara F. Management of genetic epilepsies: From empirical treatment to precision medicine. Pharmacological research 2016; 107: 426-9. 3. International League Against Epilepsy Consortium on Complex Epilepsies. Electronic address e-auea. Genetic determinants of common epilepsies: a meta-analysis of genome-wide association studies. The Lancet Neurology 2014; 13(9): 893-903. 4. Jehi L, Wyllie E, Devinsky O. Epileptic encephalopathies: Optimizing seizure control and developmental outcome. Epilepsia 2015; 56(10): 1486-9. 5. Shao LR, Stafstrom CE. Pediatric Epileptic Encephalopathies: Pathophysiology and Animal Models. Seminars in pediatric neurology 2016; 23(2): 98-107. 6. Auvin S, Cilio MR, Vezzani A. Current understanding and neurobiology of epileptic encephalopathies. Neurobiology of disease 2016; 92(Pt A): 72-89. 7. Sisodiya SM. Genetic screening and diagnosis in epilepsy? Current opinion in neurology 2015; 28(2): 136-42. 8. Villa C, Combi R. Potassium Channels and Human Epileptic Phenotypes: An Updated Overview. Frontiers in cellular neuroscience 2016; 10: 81. 9. Kaplan DI, Isom LL, Petrou S. Role of Sodium Channels in Epilepsy. Cold Spring Harbor perspectives in medicine 2016; 6(6). 10. Mantegazza M, Rusconi R, Scalmani P, Avanzini G, Franceschetti S. Epileptogenic ion channel mutations: from bedside to bench and, hopefully, back again. Epilepsy research 2010; 92(1): 1-29. 10
11. Warman Chardon J, Beaulieu C, Hartley T, Boycott KM, Dyment DA. Axons to Exons: the Molecular Diagnosis of Rare Neurological Diseases by Next-Generation Sequencing. Current neurology and neuroscience reports 2015; 15(9): 64. 12. Moller RS, Dahl HA, Helbig I. The contribution of next generation sequencing to epilepsy genetics. Expert review of molecular diagnostics 2015; 15(12): 1531-8. 13. Guerreiro R, Bras J, Hardy J, Singleton A. Next generation sequencing techniques in neurological diseases: redefining clinical and molecular associations. Human molecular genetics 2014; 23(R1): R47-53. 14. Jiang T, Tan MS, Tan L, Yu JT. Application of next-generation sequencing technologies in Neurology. Annals of translational medicine 2014; 2(12): 125. 15. Ream MA, Mikati MA. Clinical utility of genetic testing in pediatric drug-resistant epilepsy: a pilot study. Epilepsy & behavior : E&B 2014; 37: 241-8. 16. Kharbanda M, Tolmie J, Joss S. How to use... microarray comparative genomic hybridisation to investigate developmental disorders. Archives of disease in childhood Education and practice edition 2015; 100(1): 24-9. 17. Spreiz A, Haberlandt E, Baumann M, et al. Chromosomal microaberrations in patients with epilepsy, intellectual disability, and congenital anomalies. Clinical genetics 2014; 86(4): 361-6. 18. Olson H, Shen Y, Avallone J, et al. Copy number variation plays an important role in clinical epilepsy. Annals of neurology 2014; 75(6): 943-58. 19. Striano P, Coppola A, Paravidino R, et al. Clinical significance of rare copy number variations in epilepsy: a case-control survey using microarray-based comparative genomic hybridization. Archives of neurology 2012; 69(3): 322-30. 20. Copy number variant analysis from exome data in 349 patients with epileptic encephalopathy. Annals of neurology 2015; 78(2): 323-8. 21. Bonuccelli A, Valetto A, Orsini A, et al. Maternally derived 15q11.2-q13.1 duplication in a child with Lennox-Gastaut-type epilepsy and dysmorphic features: Clinical-genetic characterization of the family and review of the literature. American journal of medical genetics Part A 2016. 22. Mefford HC, Muhle H, Ostertag P, et al. Genome-wide copy number variation in epilepsy: novel susceptibility loci in idiopathic generalized and focal epilepsies. PLoS genetics 2010; 6(5): e1000962. 23. de Kovel CG, Trucks H, Helbig I, et al. Recurrent microdeletions at 15q11.2 and 16p13.11 predispose to idiopathic generalized epilepsies. Brain : a journal of neurology 2010; 133(Pt 1): 2332. 24. Stuppia L, Antonucci I, Palka G, Gatta V. Use of the MLPA assay in the molecular diagnosis of gene copy number alterations in human genetic diseases. International journal of molecular sciences 2012; 13(3): 3245-76. 25. Myers CT, Mefford HC. Advancing epilepsy genetics in the genomic era. Genome medicine 2015; 7: 91. 26. Kadalayil L, Rafiq S, Rose-Zerilli MJ, et al. Exome sequence read depth methods for identifying copy number changes. Briefings in bioinformatics 2015; 16(3): 380-92. 27. Minassian BA, Striano P, Avanzini G. Progressive Myoclonus Epilepsy: The GeneEmpowered Era. Epileptic disorders : international epilepsy journal with videotape 2016; 18(S2): 1-2. 28. Thomas RH, Berkovic SF. The hidden genetics of epilepsy-a clinically important new paradigm. Nature reviews Neurology 2014; 10(5): 283-92. 29. Parrini E, Marini C, Mei D, et al. Diagnostic Targeted Resequencing in 349 Patients with Drug-Resistant Pediatric Epilepsies Identifies Causative Mutations in 30 Different Genes. Human mutation 2016. 30. Wang J, Lin ZJ, Liu L, et al. Epilepsy-associated genes. Seizure 2017; 44: 11-20.
11
31. Mantegazza M RR, Cestèle S. Mutations of Ion Channels in Genetic Epilepsies. Epilepsy Towards the Next Decade - New Trends and Hopes in Epileptology - Springer Science+Business Media 2015: 15-34. 32. de Lera Ruiz M, Kraus RL. Voltage-Gated Sodium Channels: Structure, Function, Pharmacology, and Clinical Indications. Journal of medicinal chemistry 2015; 58(18): 7093-118. 33. Camfield P, Camfield C. Febrile seizures and genetic epilepsy with febrile seizures plus (GEFS+). Epileptic disorders : international epilepsy journal with videotape 2015; 17(2): 124-33. 34. Djemie T, Weckhuysen S, von Spiczak S, et al. Pitfalls in genetic testing: the story of missed SCN1A mutations. Molecular genetics & genomic medicine 2016; 4(4): 457-64. 35. Brunklaus A, Zuberi SM. Dravet syndrome--from epileptic encephalopathy to channelopathy. Epilepsia 2014; 55(7): 979-84. 36. Scalmani P, Rusconi R, Armatura E, et al. Effects in neocortical neurons of mutations of the Na(v)1.2 Na+ channel causing benign familial neonatal-infantile seizures. The Journal of neuroscience : the official journal of the Society for Neuroscience 2006; 26(40): 10100-9. 37. Syrbe S, Zhorov BS, Bertsche A, et al. Phenotypic Variability from Benign Infantile Epilepsy to Ohtahara Syndrome Associated with a Novel Mutation in SCN2A. Molecular syndromology 2016; 7(4): 182-8. 38. Schwarz N, Hahn A, Bast T, et al. Mutations in the sodium channel gene SCN2A cause neonatal epilepsy with late-onset episodic ataxia. Journal of neurology 2016; 263(2): 334-43. 39. Gardella E, Becker F, Moller RS, et al. Benign infantile seizures and paroxysmal dyskinesia caused by an SCN8A mutation. Annals of neurology 2016; 79(3): 428-36. 40. Anand G, Collett-White F, Orsini A, et al. Autosomal dominant SCN8A mutation with an unusually mild phenotype. European journal of paediatric neurology : EJPN : official journal of the European Paediatric Neurology Society; 20(5): 761-5. 41. Meisler MH, Helman G, Hammer MF, et al. SCN8A encephalopathy: Research progress and prospects. Epilepsia 2016; 57(7): 1027-35. 42. Galanopoulou AS. Mutations affecting GABAergic signaling in seizures and epilepsy. Pflugers Arch 2010; 460(2): 505-23. 43. Nava C, Dalle C, Rastetter A, et al. De novo mutations in HCN1 cause early infantile epileptic encephalopathy. Nature genetics 2014; 46(6): 640-5. 44. Allen NM, Mannion M, Conroy J, et al. The variable phenotypes of KCNQ-related epilepsy. Epilepsia 2014; 55(9): e99-105. 45. Thomas RH, Zhang LM, Carvill GL, et al. CHD2 myoclonic encephalopathy is frequently associated with self-induced seizures. Neurology 2015; 84(9): 951-8. 46. Galizia EC, Myers CT, Leu C, et al. CHD2 variants are a risk factor for photosensitivity in epilepsy. Brain : a journal of neurology 2015; 138(Pt 5): 1198-207. 47. Trivisano M, Pietrafusa N, Ciommo V, et al. PCDH19-related epilepsy and Dravet Syndrome: Face-off between two early-onset epilepsies with fever sensitivity. Epilepsy research 2016; 125: 32-6. 48. Balestrini S, Sisodiya SM. Pharmacogenomics in epilepsy. Neuroscience letters 2017. 49. Wallace A, Wirrell E, Kenney-Jung DL. Pharmacotherapy for Dravet Syndrome. Paediatric drugs 2016; 18(3): 197-208. 50. Wirrell EC. Treatment of Dravet Syndrome. The Canadian journal of neurological sciences Le journal canadien des sciences neurologiques 2016; 43 Suppl 3: S13-8. 51. Dalic L, Mullen SA, Roulet Perez E, Scheffer I. Lamotrigine can be beneficial in patients with Dravet syndrome. Developmental medicine and child neurology 2015; 57(2): 200-2. 52. Andrade DM. Dravet syndrome, lamotrigine, and personalized medicine. Developmental medicine and child neurology 2015; 57(2): 118-9. 53. Boerma RS, Braun KP, van den Broek MP, et al. Remarkable Phenytoin Sensitivity in 4 Children with SCN8A-related Epilepsy: A Molecular Neuropharmacological Approach. Neurotherapeutics 2016; 13(1): 192-7. 12
54. Vezyroglou K, Cross JH. Targeted Treatment in Childhood Epilepsy Syndromes. Current treatment options in neurology 2016; 18(6): 29. 55. Lee HH, Hur YJ. Glucose transport 1 deficiency presenting as infantile spasms with a mutation identified in exon 9 of SLC2A1. Korean journal of pediatrics 2016; 59(Suppl 1): S29S31. 56. Ramm-Pettersen A, Nakken KO, Haavardsholm KC, Selmer KK. Occurrence of GLUT1 deficiency syndrome in patients treated with ketogenic diet. Epilepsy & behavior : E&B 2014; 32: 76-8. 57. Gumus H, Bayram AK, Kardas F, et al. The Effects of Ketogenic Diet on Seizures, Cognitive Functions, and Other Neurological Disorders in Classical Phenotype of Glucose Transporter 1 Deficiency Syndrome. Neuropediatrics 2015; 46(5): 313-20. 58. Akman CI, Yu J, Alter A, Engelstad K, De Vivo DC. Diagnosing Glucose Transporter 1 Deficiency at Initial Presentation Facilitates Early Treatment. The Journal of pediatrics 2016; 171: 220-6. 59. van Karnebeek CD, Tiebout SA, Niermeijer J, et al. Pyridoxine-Dependent Epilepsy: An Expanding Clinical Spectrum. Pediatric neurology 2016; 59: 6-12. 60. Coci EG, Codutti L, Fink C, et al. Novel homozygous missense mutation in ALDH7A1 causes neonatal pyridoxine dependent epilepsy. Molecular and cellular probes 2016. 61. Baumgart A, Spiczak S, Verhoeven-Duif NM, et al. Atypical vitamin B6 deficiency: a rare cause of unexplained neonatal and infantile epilepsies. Journal of child neurology 2014; 29(5): 7047. 62. Jaeger B, Abeling NG, Salomons GS, et al. Pyridoxine responsive epilepsy caused by a novel homozygous PNPO mutation. Molecular genetics and metabolism reports 2016; 6: 60-3. 63. Dibbens LM, de Vries B, Donatello S, et al. Mutations in DEPDC5 cause familial focal epilepsy with variable foci. Nature genetics 2013; 45(5): 546-51. 64. Sadowski K, Kotulska-Jozwiak K, Jozwiak S. Role of mTOR inhibitors in epilepsy treatment. Pharmacol Rep 2015; 67(3): 636-46. 65. Jeong A, Wong M. mTOR Inhibitors in Children: Current Indications and Future Directions in Neurology. Current neurology and neuroscience reports 2016; 16(12): 102. Figure Caption
13
Figure 1: Example of diagnostic algorithm for genetic epilepsies.
14
Table 1. Diagnostic tests for epilepsy patients. Diagnostic test
Description
When to use it
Identify single nucleotide polymorphisms (SNP arrays) or to determine chromosomal
Epilepsy with developmental delay,
rearrangements submicroscopic (array-CGH) as
dysmorphism, ASD
Array-CGH
CNVs.
Single gene
Detects changes in the gene and if it causes amino
sequencing
acid alterations
Suspected single-gene defect (e.g. SLC2A1 in Glut-1 deficiency)
Duplication Suspicious of a single gene defect deletion of a
CNV of a single gene when sequencing is inconclusive
single gene Research of a
On parents to understand if an Sequencing of a specific mutation
specific mutation
unknown mutation is pathological
Targeted-
Sequencing and duplication/deletion research of a
resequencing
gene panel for a specific disease
disease with more genes involved
Fluorescent in situ Probes that analyse specific chromosome’s hybridization
Confirmation of a duplication/deletion portions
(FISH) Analysis of all chromosome for big
Patients with dysmorphism and/or
duplication/deletion
multiorgan dysfunction
Sequencing of all DNA only for codifying regions
Suspected genetic aetiology with
(exons) or all regions (genome)
otherwise normal investigations
Karyotype
Whole-exome and genome sequencing
15
Table 2. Examples of precision medicine for genetic epilepsies. Gene
Epilepsy syndrome
Possible treatment(s)
SCN1A
Dravet syndrome
Avoid sodium channel blockers
Early-onset epileptic
Sodium channel blockers
encephalopathy
(carbamazepine, phenytoin)
SCN8A
KCNQ2
Early-onset epileptic encephalopathy
Potassium channel openers (retigabine) or sodium channel blockers (carbamazepine) Potassium channel openers
KCNT1
Migrating partial epilepsy of
(quinidine)
infancy
NMDA (N-methyl-D-aspartate) GRIN2A
SCL2A1 TSC1/TSC2 ALDH7A1
Early-onset epileptic
receptor antagonists
encephalopathy
(memantine)
GLUT-1 deficiency
Ketogenic Diet
Tuberous sclerosis complex Pyridoxine dependency
16
Rapamicin and analogs B6 vitamin