From Molecules to Cells, Networks, and Seizures: How Does a Gene Cause Epilepsy?

Transcription

1 From Molecules to Cells, Networks, and Seizures: How Does a Gene Cause Epilepsy? Belinda Oyinkan Marquis, MD State University of New York Downstate Medical Center, Brooklyn, New York Abstract The genetics of epilepsy are evolving rapidly, with each discovery potentially having therapeutic implications. The field has developed from the miracle of gene discovery to an understanding of the functional impact of genetic changes and variability to a new therapeutic, scientific, and clinical perspective of epilepsy. At a symposium held during the 66 th Annual Meeting of the American Epilepsy Society, experts on the genetic road map of epilepsy and related medical conditions discussed the discovery of genes associated with different types of epilepsy, their variability, and their impact on clinical practice. T he genetic spectrum of epilepsy is evolving at a breakneck pace, with scientists and clinicians continually reporting new findings that may affect the everyday practice of neurologists who diagnose and treat patients with seizures. At a symposium held during the 66 th Annual Meeting of the American Epilepsy Society, speakers addressed the process of gene discovery (ie, how mutations and chromosome deletions and duplications are identified in populations and single individuals), genetic variability (ie, how the same mutation can be responsible for different phenotypes), the management of patients with a genetically related seizure disorder, and the provision of specific information and counseling for patients diagnosed with certain mutations or chromosome defects. n Gene Discovery in Epilepsy Based on a presentation by Heather C. Mefford, MD, PhD, Assistant Professor of Pediatrics, Division of Genetic Medicine, University of Washington, and Attending Physician, Medical Genetics Clinic, Seattle Children s Hospital, Seattle, Washington New methods of genomic analysis have broadened our understanding of the human genome. The emergence of genome-scanning technologies has uncovered an unexpectedly large portion of structural variations in the genome. These microscopic and submicroscopic variants include deletions, duplications, insertions, inversions, and translocations, which are collectively known as copy number variants (CNVs). 1 These CNVs are microdeletions or microduplications of segments of the genome that range from a few hundred base pairs to several hundred megabases (Mb). A change in copy number is revealed by comparing two or more genomes. 1 CNVs play an important role in genetically complex epilepsies that can either be inherited or occur de novo. 2 There are two classes of CNVs: recurrent and nonrecurrent. Recurrent CNVs often are de novo mutations, individually more frequent, and the cause of some known microdeletions. Three large, recurrent microdeletions at 15q13.3, 16p13.11, and 15q11.2 are each present in 0.5% 1% of patients with epilepsy. 3 Microdeletions of 15q13.3 are associated with idiopathic generalized epilepsies (odds ratio, 68). 4 These microdeletions are also established risk factors for related disorders, which include intellectual disability, autism, and schizophrenia. 5 Known medical conditions related to microdeletion disorders are Angelman, Prader-Willi, and Williams-Beuren syndromes. However, rare, nonrecurrent CNVs are also important. Nonrecurrent CNVs occur throughout the genome and are not sequence-dependent. Large (> 2 Mb), rare CNVs, including the deletion at 16p13.11, are enriched in patients with diverse epilepsy syndromes. 6 Rare CNVs also are found in 10% of patients with various types of epilepsy 3 and 8% of patients with epileptic encephalopathies. 7 Laboratory Methods The development of new experimental and computational strategies, such as comparative genome hybridization (CGH) and single-nucleotide polymorphism (SNP) arrays, has allowed human structural genetic variations to be analyzed at a higher resolution. 1 CGH, the most robust method for performing genome-wide scans to find novel CNVs, uses labeled fragments from a genome of interest (proband). These fragments are hybridized with a second differentially labeled genome to arrays Dr. Marquis is a Clinical Neurophysiology Fellow (Epilepsy) at the State University of New York Downstate Medical Center, Brooklyn, New York. 6 T H E N E U R O L O G Y R E P O R T V o l u m e 5 N u m b e r 3

2 that are spotted with cloned DNA fragments to reveal copy number differences between the two genomes. 1 CGH has revealed many more CNVs in humans than previously recognized. 8 SNP arrays are DNA arrays that detect polymorphisms within a population. An array contains the target nucleic acid sequence. One or more labeled allelespecific oligonucleotide (ASO) probes are applied, and a detection system that records and interprets the hybridization signal is used. 9 SNP arrays are useful for detecting unilateral disomy and consanguinity. Trio analysis Sequence mother + father + child Look for de novo change in child Severe, de novo disorders Family analysis Sequence selected individuals Use inheritance pattern Recessive, dominant, X-linked pattern Multiple, unrelated, affected individuals Sequence multiple individuals Look for mutations in the same gene across multiple affected individuals Modes of Analysis CNV analysis provides new opportunities to examine the range of genetic variations associated with disease. They point to novel candidate genes that may be used for targeted sequencing in large epilepsy cohorts. Further testing with array CGH studies is warranted for patients who have epileptic encephalopathy and epilepsy plus syndromes: epilepsy in addition to brain malformations, intellectual disability, autism spectrum disorder, or congenital anomalies. Gene-sequencing analysis has improved by significant leaps over the past 30 years. The classic Sanger sequencing method involves a DNA primer, DNA polymerase, and deoxynucleopeptides. Sample DNA is denatured and copied by polymerase chain reaction, sequenced, and then analyzed. However, this method is time-consuming and expensive. The Sanger sequencing method has been supplanted by next-generation sequencing, which can analyze millions of fragments of DNA simultaneously at significantly less expense. This method captures exomes, which are the part of the genome formed by exons; these coding portions of expressed genes provide the genetic blueprint used in the synthesis of proteins and other functional gene products. This information-rich extraction is then run through automated next-generation sequencing methods. Gene discovery is accomplished using three clinical strategies: (1) trio analysis, (2) family analysis, and (3) FIGURE 1 Three strategies for gene discovery. Adapted from a slide presented by Heather Mefford, MD, PhD. No answers Interesting phenotype Interesting family Good insurance Sequence whole exome Many possible causative genes analysis of multiple, unrelated affected individuals (Figure 1). Trio analysis involves sequencing of the mother and father of an affected child to investigate a de novo mutation in the child. Family analysis involves sequencing selected individuals within a family to establish a pattern of inheritance (ie, recessive, dominant, X-linked). Analysis of multiple unrelated affected individuals looks for mutations in the same gene. Exome sequencing for diagnosis is moving into the clinical setting rapidly. As the technology advances, its cost will continue to fall, and the technique will become more accessible. Next-generation gene panels currently Suspect a specific diagnosis? Nonspecific, epilepsyplus syndrome* Order a gene-panel analysis Test for a specific gene Dravet syndrome: SCN1A Rett syndrome: MECP2 Search for copy number variants * Epilepsy in addition to brain malformations, intellectual disability, autism spectrum disorder, or congenital anomalies FIGUre 2 Pathways to a diagnosis using exome sequencing. Adapted from a slide presented by Heather Mefford, MD, PhD. are available commercially to sequence many genes, although they are not allinclusive. Therefore, clinicians who use these tools must focus on a known disease or set of genes. If a specific diagnosis is suspected, it is recommended that clinicians investigate a particular gene (eg, SCN1A for Dravet syndrome, MECP2 for Rett syndrome). In cases of a nonspecific epilepsy-plus syndrome or multiple causative genes, CNV testing or gene-panel analysis should be considered. If an interesting phenotype and a supportive family history are involved, whole-exome sequencing can be considered if it is financially feasible (Figure 2). T H E N E U R O L O G Y R E P O R T W i n t e r

3 n How Do We Determine the Functional Impact of Genetic changes? Based on a presentation by Jack Parent, MD, Associate Professor of Neurology and Co-Director, Comprehensive Epilepsy Program, University of Michigan Medical School, Ann Arbor, Michigan The growing advances in genetic sequencing broaden the opportunity to discover possible disease-causing mutations. The key elements to consider when evaluating suspected mutations and variants include changes in an amino-acid sequence made by the mutation, evolutionary conservation of the altered sequence, and the effects on protein function or structure resulting from the variation. After a gene is determined to be pathogenic, the next step is to determine how a particular mutation functions and leads to epilepsy. The effects of gene mutations on proteins are manifested in various ways. Function may be lost when a protein either does not engage in its native function or is degraded. This commonly occurs with autosomal-recessive or autosomal-dominant haploinsufficiency, in which quantitative reduction of gene expression to 50% of normal levels results in an abnormality. 10 Conversely, function may be gained when a normal novel protein has a de novo or toxic new function. Finally, there are dominant-negative mutations, in which the mutant protein suppresses some other normal protein. Dravet syndrome, or severe myoclonic encephalopathy of infancy (SMEI), is a prime example of mutations leading to epilepsy. This epileptic encephalopathy is related to early-onset seizures and is associated with cognitive impairments and 15% mortality by adolescence. 11 Most often it is caused by a de novo loss of function mutation in the neuronal sodiumchannel gene SCN1A, which leads to haploinsufficiency of NaV1.1 channels. 12 Voltage-gated sodium channels have critical roles in the initiation and propagation of action potentials and are crucial regulators of neuronal excitability. Mutations in the NaV1.1 channel gene SCN1A cause genetically distinct epilepsy syndromes. Evaluating Cause and Effect The functional effects of a mutation must be tested by creating cell-culture models. Heterologous expression systems can be created with the use of Xenopus oocytes, human embryonic kidney cells, or other progenitor cells. These systems are used to express mutant human genes and are easily studied using electrophysiologic methods. Further, they may be used for protein expression and targeting, enzyme assays, and defined protein interactions. However, heterologous expression does not mimic the effects in neurons or in vivo. 13 Primary neuron cultures are advantageous, because they allow affected neuron cells to be evaluated as they function. However, they are technically more difficult to cultivate; their expression cannot be well controlled; and, without being in a native environment, the cells may change in culture. Transgenic knockout mouse models showed that mice with a heterozygous functioning Scn1a +/ gene (Dravet syndrome model) exhibited a reduced sodium current in interneurons. 14 These mouse models were unique in that the phenotype of the Scn1a +/ mice phenocopied human SMEI. The finding that haploinsufficiency of a sodium channel causes epilepsy was unsuspected, because reduced sodium current could lead to inexcitability rather than hyperexcitability. The theory of an alternate effect underlying the causation of epilepsy is based upon loss of sodium current in hippocampal inhibitory interneurons able to transmit or secrete γ-aminobutyric acid (GABA). Intractable epilepsy could result from failure in the excitability of hippocampal GABAergic inhibitory neurons. 14 There are, however, limitations to the value of transgenic mouse models. Murine models do not harbor a patient s other genetic variations, and there can be variability in the breeding of transgenic mice. In addition, gene expression may vary among different lines of transgenic mice. Induced pluripotent stem cell (ipsc) technology offers enormous potential for understanding disease states by creating models of the genetic milieu on a microscopic level. The technique involves direct reprogramming of somatic cells to a stem cell (pluripotent state). The ipscs can then be modified genetically and differentiated into relevant tissue to be examined for disease mechanisms or to screen for new therapies. Dravet syndrome can be better understood with ipscs. As an example, imagine two subjects with Dravet syndrome one harbored an SCN1A splice site mutation and the other, an SCN1A nonsense mutation. After ipscs derived from Dravet syndrome patient fibroblasts were compared with those of controls, Dravet ipsc neurons showed increased sodium current and increased excitability, firing repetitively and bursting spontaneously. ipscs also have been created to study sudden unexplained death of epileptic patients (SUDEP). Ion channelopathies likely predispose patients to SUDEP. 11 Individuals with Dravet syndrome and generalized epilepsy with fever symptoms plus another SCN1A channelopathy have a high incidence of SUDEP. The Nav1.1 subunit is expressed in cardiac myocytes. In work not yet published, Dravet syndrome patient fibroblasts were reprogrammed to ipscs and then modified and differentiated to become cardiac myocytes; the same was performed with controls. Cardiac myocytes derived from the ipscs of controls displayed organized and uniform contractility. Cardiac myocytes derived from Dravet syndrome ipscs displayed faster and irregular myocyte contraction. Electrophysiologic studies showed increased sodium currents in cardiac myocytes from Dravet syndrome patients and mutant mice, and the altered sodium currents affected cardiomyocyte potential, leading to an arrhythmogenic state. However, ipscs have limitations. Although the cells may be genetically manipulated for reprogramming to avoid genetic integration, learning how to make regional and subtype-specific neurons is still a challenge, since incomplete maturation and abnormal growth may result. Assessing ipsc-derived neurons in vivo by transplanting ipsc neurons from Dravet syndrome patients and controls into a rat hippocampus and then taking brain slices 8 T H E N E U R O L O G Y R E P O R T V o l u m e 5 N u m b e r 3

4 for hippocampal recording in a natural environment may overcome this problem. With cell culture and animal models, the key is to identify and confirm potential epilepsy-causing mutations. The patientspecific models of epilepsy mutations provide abundant knowledge and allow future researchers to set a new goal of creating personalized therapy. With multiple models of genetic epilepsies, it may be possible to perform drug screening, to test existing therapies, and to analyze new therapies and toxicities in the future. n what Can We Learn About Epilepsy from Genome Sequences? Based on a presentation by David Goldstein, PhD, Richard and Pat Johnson Distinguished University Professor, Director of the Center for Human Genome Variation, and Professor of Molecular Genetics and Microbiology and Biology, Duke University School of Medicine, Durham, North Carolina Genetic mechanisms that influence the development of epilepsy in the vast majority of patients remain unknown. Gene Identification in Complex Epilepsies Historically, gene-identification studies have been performed using linkage studies and association approaches. Linkage studies draw upon genetic and phenotypic information from multiple generations to map regions of the genome that are co-inherited with familial disease. Regions of interest from linkage studies tend to be relatively large due to the few opportunities for recombination within the families investigated. Association studies investigate variants at individual loci that occur more commonly in unrelated patients than controls. Different unrelated individuals with a unique recombination history are evaluated; therefore, the size of the associated region theoretically should be much smaller, and specific susceptibility genes may be more easily identified than possible with a linkage study. To identify specific susceptibility genes with a particular linkage, linkage studies are often followed by association mapping. 15 Candidate-gene association studies are the next step in gene identification. These studies focus on genes likely to be involved in disease pathophysiology. Candidate-gene studies in patients with epilepsy have been less than successful, partially because of inadequate knowledge of disease pathophysiology needed to select candidate genes. As a result, genome-wide association studies (GWAS) have been employed to understand variants. GWAS use genotyping chips to tag variants that may be common in a population. 16 GWAS have been useful for identifying secure risk factors for common diseases. However, GWAS have failed to identify many variants of large effect in complex diseases such as epilepsy. Consequently, researchers have a blossoming interest in the possibility that variants too rare to Rare variants, such as single-nucleotide, insertion, and/or deletion variants, may be important contributors to complex diseases such as epilepsy. be represented well on GWAS chips may be important contributors to common diseases, including the epilepsies. This has led to considerable interest in the use of next-generation sequencing, which allows for the near-comprehensive characterization of genetic variants across the genome to identify rare risk factors for disease. 17 The rare-variant hypothesis is supported by the recent observation that rare CNVs can have large effects on neuropsychiatric disease risk. 18 This observation, along with the fact that only 9% of variants reported as disease-associated in the Human Gene Mutation Database are CNVs, 19 strongly suggests that other types of rare variants, such as single-nucleotide, insertion, and/or deletion variants, may be important contributors to complex diseases. Next-generation sequencing has great potential to identify susceptibility genes for complex diseases; however, large sample sizes will be required, because locus and allelic heterogeneity are predicted to be high, and all genomes carry a large number of rare functional variants. 20 The Epi4K Consortium The National Institute of Neurological Disorders and Stroke (NINDS) has recognized the importance of accelerating progress in this area of epilepsy research. In 2010, the NINDS issued a Funding Opportunity Announcement for the creation of the Epi4K Consortium, 21 a center without walls to focus on the genetics of human epilepsy. This collaborative study aims to sequence at least 4,000 subjects with epilepsy. The geographically boundless structure consists of three cores and four scientific projects, as well as a steering committee made up of primary investigators and representatives from NINDS. Three projects will analyze specific sets of epilepsy cohorts (namely, epileptic encephalopathies, multiplex families and pairs, and prognosis) drawn from seven large-scale genetic studies conducted around the world, and one will apply cutting-edge analytic techniques related to the detection of CNVs. Project 1 of Epi4K addresses the genetics of epileptic encephalopathies that are refractory to medication and associated with comorbid cognitive dysfunction and behavioral disturbances. The two more common types are infantile spasm and Lennox-Gastaut syndrome. Infantile spasm is the most common of the epileptic encephalopathies, occurring in 1 in 3,000 live births and becoming noticeable at 4 12 months of age. 22 This syndrome is associated with the characteristic electroencephalographic pattern of hypsarrhythmia, which is the sine qua non of the syndrome. Spasms are associated with an electrodecremental response. Approximately 50% 60% of infantile spasm cases are associated with developmental brain malformations, tuberous sclerosis complex, chromosomal syndromes, or metabolic conditions. Many patients with infantile spasm or other early-onset epileptic encephalopathies may experi- T H E N E U R O L O G Y R E P O R T W i n t e r

5 ence an evolution to Lennox-Gastaut syndrome, suggesting a likely shared etiology. 23 Lennox-Gastaut syndrome typically is noticed at 1 8 years of age and is characterized by mixed seizure types (tonic, atonic, myoclonic, atypical absence, focal, and generalized) and intellectual disabilities. 24 Its cause is unknown in about 25% 35% of cases; the others are symptomatic of structural or metabolic abnormalities. Participants in Project 1 are being contributed by the Epilepsy Phenome/ Genome Project (EPGP), 25 an actively enrolling, 5-year, NINDS-funded study that will involve 5,250 members of families with epilepsy. Participants will undergo detailed phenotyping and analysis of medical records, electroencephalography, and magnetic resonance imaging. All Project 1 samples are from the infantile spasm/lennox-gastaut syndrome arm of the EPGP. In preliminary results obtained from whole exome sequencing of 165 trios (75 infantile spasm and 90 Lennox-Gastaut syndrome probands and parents) to identify new genes and pathways conferring risk of epileptic encephalopathies, there were 180 de novo mutations enriched in specific gene sets. These mutations included 14 causal de novo mutations in six known genes for epileptic encephalopathy (SCN1A, SCN8A, STXBP1, SCN2A, CDKL5, and KCNQ2). Of these 14 mutations, 13 were newly identified as causal. Projects 2 4. The objective of Project 2 is to identify genomic variations that influence risk for common forms of idiopathic generalized epilepsies and nonlesional focal epilepsies found in families with two or more affected members. The objective of Project 3 is to explore the relationship between genetic variation and prognosis. The Project 4 objective is to apply novel computational algorithms to next-generation sequencing data to detect epilepsy-associated CNVs. The goal of the Epi4K Consortium is to emphasize careful phenotyping of subjects and to increase accessibility to next-generation sequencing, state of-the-art methods for genomic analysis, and the advantages of large-scale collaborations. If successful, this collaborative process will advance the identification of the causes of many forms of so-called idiopathic and cryptogenic epilepsy, which will drive the development of drugs used to treat epilepsy and, possibly, disease-modifying or antiepileptogenic agents. This study also opens doors for understanding the biologic bases of related genetic risks and assessing their translational implications. 15 n How Can Genetic Information Impact Management? Based on a presentation by Samuel Berkovic, MD, FRS, Laureate Professor of Medicine, University of Melbourne, Melbourne, Victoria, Australia The genetic understanding of epilepsy causation continues to expand with continued advances in technology and research. Clinicians must now learn to apply the information and decide how it will influence the management of patients with epilepsy. Traditionally, neurologists have believed that after congenital cortical malformations, trauma, and stroke, idiopathic forces are the cause of approximately 75% of epilepsy cases. 26 Until recently, familial aggregation studies, twin studies, and multiplex family studies have been the sources of gene discovery related to epilepsy. Familial aggregation studies compare the frequency of a disease in particular relatives to controls and determine the recurrence risk ratio. The overall recurrence risk ratio for epilepsy is 2.5 in first-degree relatives, 4 9 for generalized epilepsy, 2 3 for partial epilepsy, and 3 5 for febrile seizures, suggesting that there is a large degree of genetic inheritance in particular for generalized epilepsies. Twin studies support this premise. Vadlamudi et al 27 evaluated a cohort of twins to examine the genetics of epilepsy syndromes. In all, 418 twin pairs were studied, with one or both of each pair reporting seizures. More monozygous pairs were concordant for seizures than were dizygous pairs. When analyzed according to epilepsy syndrome, the case-wise concordances for generalized epilepsies (99 patients; monozygous, 0.73; dizygous, 0.33) were greater than were those for partial epilepsies (103 patients; monozygous, 0.34; dizygous, 0.04), with intermediate values for febrile seizures (180 patients; monozygous, 0.60; dizygous, 0.14) and unclassified epilepsies (36 patients; monozygous, 0.43; dizygous, 0.13). The investigators concluded that genetic factors are particularly important in the generalized epilepsies, but they also play a role in the partial epilepsies. In addition, the high frequency of concordant monozygous pairs having the same major syndrome strongly suggests there are syndrome-specific genetic determinants, rather than a broad genetic predisposition, to seizures. 28 Multiplex family studies select for familial units having two or more affected members. Many gene discoveries, such as autosomal-dominant nocturnal frontal-lobe epilepsy, have resulted from these studies. Practical Use of These Discoveries The growing precision in gene identification has advanced the understanding of epilepsy causation. Epilepsies formerly broadly classified as being idiopathic in nature now are being identified as genetic in origin. Monogenic epilepsies often are related to dominant inheritance and can display genetic heterogeneity with pleiotropic expression. In other words, one gene can influence many phenotypic traits. Such genes can cause ion-channel subunit abnormalities, such as sodium or potassium voltage-gated channels or ligand-gated channels (eg, nicotinic or GABA-associated channels). In addition, causative monogenic non ion-channel genes have been identified. Some examples are the LGI1 gene associated with temporal lobe epilepsy, the GLUT1 gene associated with early-onset absence seizures, and the PRRT2 gene associated with a spectrum of neurologic disorders, including benign familial infantile epilepsy, febrile seizures, childhood absence seizures, paroxysmal dyskinesias, migraines, and hemiplegic migraines. 29 Complex epilepsies have been associated with common variants 10 T H E N E U R O L O G Y R E P O R T V o l u m e 5 N u m b e r 3

6 and rare variants associated with CNVs. De novo mutagenesis has unexpectedly shown significant importance in various epilepsies, particularly Dravet syndrome. On the basis of a study of monozygotic twins showing discordant inheritance of SCN1A, 30,31 the timing of de novo mutagenesis is linked to parental germ-line mutations, postzygotic mutations, and mosaicism. In this study, most cases of the de novo mutation occurred in the parental germ line, giving rise to a mutated sperm or egg. Mutations in SCN1A occur more frequently in the male germ line. In one set of monozygotic twins, the proband displayed the SCN1A mutation in multiple cell lines, and the twin was unaffected; this suggested that mutagenesis occurred early in postzygotic development at the two cell premorula stage. Mosaicism refers to the presence of two different cell lines after fertilization, which indicates the timing of mutagenesis. Somatic mosaicism occurs in non sex cells, whereas germline mosaicism occurs in gamete-forming cells. In Dravet syndrome, somatic mosaicism is found when mildly affected parents have one or more affected offspring. Germ-line mosaicism is inferred in cases of unaffected parents having multiple affected offspring. 27 These are important identifications for genetic counseling. Gametal mutations are associated with negligible recurrence risk, whereas a germ-line mosaicism is associated with a high risk of recurrence (Figure 3). 27,32 38 The expanding role of genetics in the daily practice of neurology and management of epilepsy patients exemplifies how important it is for neurologists to improve genetic literacy. The initial step is to obtain a family history and to make a precise diagnosis of a clinical syndrome. This information will serve as a foundation for choosing appropriate testing. Clinicians should consider performing high-yield tests in the appropriate setting when faced with a particular clinical presentation. For example, a patient presenting with epilepsy plus multiple comorbid syndromic features, such as intellectual disability, autistic spectrum disorder, congenital anomalies, or brain malformations, may Somatic mosaicism Premorula mutation Mildly affected parent; child with Dravet syndrome Frequency in Dravet syndrome unknown; discordant monozygous twins Germ-line mosaicism (male) Unaffected parent; multiple affected children Germ-line (gametal) mutation Most cases of Dravet syndrome; concordant monozygous twins FIGURE 3 Timing of mutations in Dravet syndrome. Gray boxes show the timing of mutations, and green boxes describe evidence from twin and family studies. Most cases of this syndrome are believed to be due to de novo mutations in gametes (usually paternal 31 ), although in non-twin patients with the syndrome, postzygotic mutation cannot be ruled out. Somatic mosaicism has been described in mildly affected parents (father or mother) with a child or children with Dravet syndrome Germ-line mosaicism in fathers occurs in the adult testis, whereas germ-line mosaicism in mothers (not shown) occurs in early development, because oocytes are mature by 5 months of fetal life. Germ-line mosaicism is inferred when a healthy parent has multiple affected children. 32,34 37 Reproduced, with permission, from Vadlamudi et al. 27 T H E N E U R O L O G Y R E P O R T W i n t e r

7 be tested with CGH and SNP arrays for CNVs. In patients with a clinical presentation in adolescence or adulthood that suggests GLUT1 encephalopathy, earlyonset absence epilepsy, or epilepsy and paroxysmal exercise-induced dyskinesias, SLC2A1 gene sequencing should be considered. A confirmed diagnosis can be treated with a ketogenic diet. In children presenting with signs and symptoms consistent with Dravet syndrome, sodiumchannel blockers should be avoided to prevent exacerbation of seizures. Dravet syndrome should also be considered in adults with a suggestive history; an abnormal gait can be a helpful diagnostic clue in an undiagnosed patient. A study by Rodda et al 38 showed that children with Dravet syndrome show progressive gait deterioration in the second decade of life; they tend to have a crouch gait and skeletal malalignment. 39 In families harboring a female inheritance of syndromes suggestive of Dravet syndrome that do not feature an SCN1A mutation, testing for a PCDH19 mutation may be in order. Mutations in PCDH19 (which encodes protocadherin 19 on the X chromosome) were identified as epilepsy in females with mental retardation (EFMR) or a Dravetlike phenotype. In this case, heterozygous females are affected, whereas hemizygous males are spared. 40 In daily practice, genetic diagnoses aid in determining causation and thereby avoid unnecessary testing. The results, however, may alter treatment, as in individuals having GLUT1 or SCN1A mutations. Diagnosis is essential for counseling and for discussing the risk of recurrent de novo mutations or other unusual inheritance patterns. n the Promise of Epilepsy Genetics: A Personal and Scientific Perspective Based on a presentation by Tracy Dixon-Salazar, PhD, Postdoctoral Fellow, University of California at San Diego, San Diego, California Gene identification using exome sequencing may be studied by evaluating consanguineous families or small families with many linkage peaks. Via such analysis, genomic variants are filtered Prioritize Filter Variants identified Type of mutation Sequence exome Indels, splice coding, nonsynonymous Mapping Variant calling Conservation across species FIGUre 4 Exome sequencing to evaluate consanguineous families or small families with many linkage peaks. Adapted from a slide presented by Tracy Dixon-Salazar, PhD. and prioritized to show gene mutations relevant to disease causation (Figure 4). Next-generation exome sequencing has moved from the realm of research to the clinic. It will have a significant impact on diagnosis and management, because greater knowledge of genetic disease may reduce costs, advance accuracy, and point to unsuspected, yet treatable, conditions. This hypothesis was studied by Dixon- Salazar et al, 41 who used whole-exome sequencing in 118 patients diagnosed with pediatric-onset neurodevelopmental disease; most known causes had been excluded. The authors identified 22 previously unidentified disease-causing genes (19% of the cohort) and further established exome sequencing as a useful tool for gene discovery. Exome sequencing uncovered 10 probands (8% of the cohort) having mutations in genes that were associated with a disease that was different from that initially diagnosed. Thus, exome sequencing was shown to yield a correct diagnosis. Upon further medical evaluation, these mutations accounted for each proband s disease, leading to a change in diagnosis and some associated changes in patient management. These results provided evidence that genomic strategies may clarify the diagnosis of certain patients with neurodevelopmental disorders. An example of a potentially treatable neurodevelopmental disorder that may be treated following gene identification is the BCKDK mutation, which is associated with autism. Novarino et al 42 have identified inactivating mutations in BCKDK in consanguineous families with autism, epilepsy, and intellectual disability. The encoded protein is responsible for Homozygous variants Protein damage prediction Variant filtering Genotype not in controls Relevance to disease Variant prioritization Variants in linkage intervals phosphorylation-mediated inactivation of the E1α subunit of BCKDK. Patients with homozygous BCKDK mutations display reductions in BCKDK messenger RNA and protein, E1α phosphorylation, and plasma branched-chain amino acids. Bckdk knockout mice show abnormal brain amino acid profiles and neurobehavioral deficits that respond to dietary supplementation. These findings suggest that autism presenting with intellectual disability and epilepsy caused by BCKDK mutations may represent a potentially treatable syndrome. An unpublished case study of a 19-year-old female with Lennox-Gastaut syndrome of unknown cause is a fitting example of another treatable case. She was diagnosed at 32 months of age, had been experiencing seizures per month, and was unresponsive to over 26 trials of antiepileptic therapy. Exome-sequencing analysis of this patient identified numerous high-impact variants in calcium-channel subunits. The most common genetic variant of L-type calcium channels is gain of function; therefore, the receptor may have allowed for increased calcium influx that led to increased calcium at the synapse and increased neurotransmitter release. This theory was supported by increased seizure frequency with calcium supplementation. After weighing the risk and benefits, investigators evaluated the use of off-label verapamil, a calcium-channel blocker, in this patient. Administration of verapamil reduced seizure frequency by 75% 80%. Interestingly, this patient was the daughter of the presenter and the leading example of the implications of exome sequencing on epilepsy management. 12 T H E N E U R O L O G Y R E P O R T V o l u m e 5 N u m b e r 3

8 Exome sequencing has great potential to allow greater understanding of novel disease-causing genes in genetically enriched families, identification of known causes of disease, correction of diagnosis and prognosis, and direction of treatment. Exome sequencing will change the conceptualization of epilepsy. Soon, analysis of unique de novo mutations and genes in patients with epilepsy is expected to be the rule rather than the exception. With relevant disease-causing genes, the possibility of repurposing a drug or designing a novel drug to treat a specific gene defect is plausible. This new paradigm for diagnosis and management opens the doors for the personalized treatment of patients with epilepsy. References 1. Feuk L, Carson A, Scherer S. Structural variation in the human genome. Nature. 2006;7: Scheffer IE, Berkovic SF. Copy number variants an unexpected risk factor for the idiopathic generalized epilepsies. Brain. 2010:133: 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 Genet. 2010;6:e Dibbens LM, Mullen S, Helbig I, et al. Familial and sporadic 15q13.3 microdeletions in idiopathic generalized epilepsy: precedent for disorders with complex inheritance. Hum Mol Genet. 2009;18: Mefford HC, Mulley JC. Genetically complex epilepsies, copy number variants and syndrome constellations. Genome Med. 2010;2: Heinzen EL, Radtke RA, Urban TJ, et al. Rare deletions at 16p13.11 predispose to a diverse spectrum of sporadic epilepsy syndromes. Am J Hum Genet. 2010;86: Mefford HC, Yendle SC, Hsu C, et al. Rare copy number variants are an important cause of epileptic encephalopathies. Ann Neurol. 2011;70: Mills RE, Walter K, Stewart C, et al. Mapping copy number variation by population-scale genome sequencing. Nature. 2011;470: Sherry ST, Ward MH, Kholodov M, et al. dbsnp: the NCBI database of genetic variation. Nucleic Acids Res. 2001;29: Meisler MH, Kearney JA. Sodium channel mutations in epilepsy and other neurological disorders. J Clin Invest. 2005;115: Genton P, Velizarova R, Dravet C. Dravet syndrome: the long-term outcome. Epilepsia. 2011;52: Claes L, Del-Favero J, Ceulemans B, Lagae L, Van Broeckhoven C, De Jonghe P. De novo mutations in the sodium-channel gene SCN1A cause severe myoclonic epilepsy of infancy. Am J Hum Genet. 2001;68: Escayg A, Goldin AL. Sodium channel SCN1A and epilepsy: mutations and mechanisms. Epilepsia. 2010;51: Yu FH, Mantegazza M, Westenbroek RE, et al. Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy. Nature. 2006;9: Cardon LR, Bell JI. Association study designs for complex diseases. Nat Rev Genet. 2001;2: Barrett JC, Cardon LR. Evaluating coverage of genome-wide association studies. Nat Genet. 2006;38: Cirulli ET, Goldstein DB. Uncovering the roles of rare variants in common disease through whole-genome sequencing. Nat Rev Genet. 2010;11: Need AC, Goldstein DB. Whole genome association studies in complex diseases: where do we stand? Dialogues Clin Neurosci. 2010;12: Stenson PD, Mort M, Ball EV, et al. The Human Gene Mutation Database: 2008 update. Genome Med. 2009;1: Pelak K, Shianna KV, Ge D, et al. The characterization of twenty sequenced human genomes. PLoS Genet. 2010;6:e The Epi4K Consortium. Epi4K: gene discovery in 4,000 genomes. Epilepsia. 2012;53: Frost FD, Hrachovy RA. Infantile Spasms. Boston, MA; Kluwer Academic Publishers; Osborne JP, Lux AL, Edwards SW, et al. The underlying etiology of infantile spasms (West syndrome): information from the United Kingdom Infantile Spasms Study (UKISS) on contemporary causes and their classification. Epilepsia. 2010;51: Camfield PR. Definition and natural history of Lennox-Gastaut syndrome. Epilepsia. 2011:52: Epilepsy Phenome/Genome Project Accessed December 11, Hauser WA, Kurland LT. The epidemiology of epilepsy in Rochester, Minnesota, 1935 through Epilepsia. 1975;16: Vadlamudi L, Dibbens LM, Lawrence KM, et al. Timing of de novo mutagenesis a twin study of sodium-channel mutations. N Engl J Med. 2010;363: Berkovic SF, Howell RA, Hay DA, Hopper JL. Epilepsies in twins: genetics of the major epilepsy syndromes. Ann Neurol. 1998;43: Marini C, Conti V, Mei D, et al. PRRT2 mutations in familial infantile seizures, paroxysmal dyskinesia, and hemiplegic migraine. Neurology. 2012;79: Arnheim N, Calabrese P. Understanding what determines the frequency and pattern of human germline mutations. Nat Rev Genet. 2009;10: Heron SE, Scheffer IE, Iona X, et al. De novo SCN1A mutations in Dravet syndrome and related epileptic encephalopathies are largely of paternal origin. J Med Genet. 2010;47: Gennaro E, Santorelli FM, Bertini E, et al. Somatic and germline mosaicisms in severe myoclonic epilepsy of infancy. Biochem Biophys Res Commun. 2006;341: Marini C, Mei D, Cross JH, Guerrini R. Mosaic SCN1A mutation in familial severe myoclonic epilepsy of infancy. Epilepsia. 2006;47: Marini C, Scheffer IE, Nabbout R, et al. SCN1A duplications and deletions detected in Dravet syndrome: implications for molecular diagnosis. Epilepsia. 2009;50: Depienne C, Arzimanoglou A, Trouillard O, et al. Parental mosaicism can cause recurrent transmission of SCN1A mutations associated with severe myoclonic epilepsy of infancy. Hum Mutat. 2006;27: Morimoto M, Mazaki E, Nishimura A, et al. SCN1A mutation mosaicism in a family with severe myoclonic epilepsy in infancy. Epilepsia. 2006;47: Selmer KK, Eriksson AS, Brandal K, Egeland T, Tallaksen C, Undlien DE. Parental SCN1A mutation mosaicism in familial Dravet syndrome. Clin Genet. 2009;76: Rodda JM, Scheffer IE, McMahon JM, Berkovic SF, Graham HK. Progressive gait deterioration in adolescents with Dravet syndrome. Arch Neurol. 2012;69: Jansen FE, Sadleir LG, Harkin LA, et al. Severe myoclonic epilepsy of infancy (Dravet syndrome): recognition and diagnosis in adults. Neurology. 2006;67: Genes in infantile epileptic encephalopathies. In: Depienne C, Gourfinkel-An I, Baulac S, et al, eds. Jasper s Basic Mechanisms of the Epilepsies. 4th ed. Bethesda, MD: National Center for Biotechnology Information; Dixon-Salazar TJ, Silhavy JL, Udpa N, et al. Exome sequencing can improve diagnosis and alter patient management. Sci Transl Med. 2012;4:138ra Novarino G, El-Fishawy P, Kayserili H, et al. Mutations in BCKD-kinase lead to a potentially treatable form of autism with epilepsy. Science. 2012;338: T H E N E U R O L O G Y R E P O R T W i n t e r