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CRITICAL REVIEW AND INVITED COMMENTARY Obtaining genetic testing in pediatric epilepsy *†Margie A. Ream, and *†Anup D. Patel Epilepsia, 56(10):1505–1514, 2015 doi: 10.1111/epi.13122 SUMMARY Margie Ream is an assistant professor of pediatric neurology at Nationwide Children’s Hospital and the Ohio State University in Columbus, Ohio. The steps from patient evaluation to genetic diagnosis remain complicated. We discuss some of the genetic testing methods available along with their general advantages and disadvantages. We briefly review common pediatric epilepsy syndromes with strong genetic association and provide a potentially useful algorithm for genetic testing in drug-resistant epilepsy. We performed an extensive literature review of available information as it pertains to genetic testing and genetics in pediatric epilepsy. If a genetic disorder is suspected as the cause of epilepsy, based on drug resistance, family history, or clinical phenotype, timely diagnosis may reduce overall cost, limit the diagnostic odyssey that can bring much anxiety to families, improve prognostic accuracy, and lead to targeted therapy. Interpretation of complicated results should be performed only in collaboration with geneticists and genetic counselors, unless the ordering neurologist has a strong background in and understanding of genetics. Genetic testing can play an important role in the care provided to patients with epilepsy. KEY WORDS: Exome sequencing, Next generation sequencing, Diagnosis. Epilepsy is a common neurologic disorder affecting 1–1.5% of the world’s population and is more commonly diagnosed in children than adults. Historically, up to 70% of epilepsy etiology was “idiopathic,”1 but with the advent of widely available genetic testing, an etiology in subsets of epilepsy patients (for example, generalized epilepsy with developmental delay) may be obtainable in >30%.2 Updated International League Against Epilepsy (ILAE) classification emphasizes genetic classification while downplaying the utility of the designation “idiopathic.”3 However, the steps from patient evaluation to genetic diagnosis remain complicated. Later we discuss some of the genetic testing methods available along with their general advantages and disadvantages. Then we briefly review common pediatric epilepsy syndromes with strong genetic associations and Accepted July 22, 2015; Early View publication September 8, 2015. *Nationwide Children’s Hospital, Columbus, Ohio, U.S.A.; and †The Ohio State University College of Medicine, Columbus, Ohio, U.S.A. Address correspondence to Anup D. Patel, ED 533, 700 Children’s Drive, Columbus, OH 43205, U.S.A. E-mail: [email protected] Wiley Periodicals, Inc. © 2015 International League Against Epilepsy provide a potentially useful algorithm for genetic testing (Table 1). Genetic Testing Background and Methods The human genome contains approximately 3 billion DNA bases that encode approximately 25,000 genes spread across 23 chromosomes. Pathogenic changes can occur at many levels. Two major classes of variation are seen: copy number variation (CNV) in which there are duplications or deletions of portions of DNA, and single nucleotide variant (SNV) in which a single base position has been altered. Once a sequence is determined, it is compared to a reference sequence, which is based on consensus of thousands of individuals of different ethnicities to determine if the sequence in question is normal or varies from the expected standard reference. Variants are classified by their pathogenicity, either based on previous reports or on analysis by in silico models (Fig. 1). New technology has broadened the way in which genetic variation can be evaluated. Traditional base by base 1505 1506 M. A. Ream and A. D. Patel Key Points • • • Table 1. Syndromes and some of the more commonly associated genes Syndrome Proper application of genetic testing in pediatric epilepsy requires understanding of the advantages and limitations of different testing modalities Genetic testing can be expensive, but potentially helpful Genetic counseling is helpful in consenting for testing and result interpretation sequencing (Sanger sequencing) is time consuming and targets one gene at a time but is highly accurate and generally less expensive per test ordered than newer alternatives. Next generation, or massively parallel, sequencing (NGS) allows for rapid sequencing of large numbers of DNA segments that are broken into smaller pieces, sequenced, and then realigned and analyzed computationally. NGS has made large gene panels, whole exome sequencing (WES), and even whole genome sequencing (WGS) possible. Gene panels sequence a list of genes known to be associated with a specific phenotype (e.g., X-linked intellectual disability, infantile onset epilepsy, etc.). As research increases our understanding, new genes are often added to the list. WES offers a broad evaluation for genetic variation by sequencing most of the protein-encoding exons and splice junctions in a patient’s genome, as it is not limited to a list of genes known to be associated with a phenotype. However, costs for techniques using NGS are usually high due to the labor intensive interpretation of the vast amount of data that is collected; but per gene sequenced, NGS is cheaper than Sanger sequencing. The data obtained from NGS often includes many variant of unclear significance (VUS) that require interpretation but may not aid in a diagnosis. Parental samples are often required to further classify a VUS. Whole genome sequencing evaluates most of the DNA content of the entire genome but is not available clinically at this time. NGS does not provide a panacea for genetic diagnosis. Mutations in noncoding areas and introns are not covered by NGS technology as applied to WES. Triplet repeats, as in fragile X, abnormal methylation, as in Angelman syndrome, and some large insertions, deletions, and duplications can be missed by WES. In general turn-around time for NGS is also much longer (up to 4 months) than for single gene sequencing due to the increased resources needed to interpret the results. In addition to technical difficulties, genetic testing is not without its controversy and ethical dilemmas.4 Confusion can exist in the results obtained and their implications to a patient. Ethical considerations such as paternity and discovery of genes related to diseases with later onset, such as Alzheimer’s, must be considered for WES. Once a genetic diagnosis is obtained, the family may subsequently carry a “label” and other family members may unwillingly become Epilepsia, 56(10):1505–1514, 2015 doi: 10.1111/epi.13122 Benign familial neonatal convulsions Benign familial infantile convulsions X-linked infantile spasms Glucose transporter deficiency syndrome Pyridoxine dependent seizures Pyridoxal phosphate responsive seizures Folate deficiency Dravet syndrome GEFS+ EFMR PME Focal epilepsies Epileptic encephalopathies Genes KCNQ2 KCNQ3 PRRT2 SCN2A CDKL5 ARX SLC2A ALDH1A7 PNPO FOLR SCN1A SCN2A GABRG2 GABRA1 PCDH19 STXBP1 HCN1 SCN1A SCN2A SCN1B GABA-A GABRD GABRG2 PCDH19 EMP2A/laforin EMP2B/malin cystatin B CHRNA4 CHRNA2 CHRNB2 Multiple Comments Testing provides definitive diagnosis Testing provides definitive diagnosis Testing provides definitive diagnosis Testing leads to definitive treatment Testing leads to definitive treatment Testing leads to definitive treatment Testing leads to definitive treatment Low MTHFR also seen in 3-phosphoglycerate dehydrogenase deficiency and some mitochondrial disorders Testing provides diagnosis with treatment implications Testing provides definitive diagnosis Testing provides definitive diagnosis Testing provides definitive diagnosis with some treatment implications Testing provides definitive diagnosis Testing proves definitive diagnosis and allows for prognosis This list is not exhaustive and is frequently added to by new literature. aware of their potential carrier status. Families should meet with a genetic counselor prior to ordering WES and often before ordering large panels. Genetic counseling is also usually required once results are available to help interpret and apply results to specific patients. Many testing companies also have genetic counselors available to discuss results with ordering providers. It is important to note that genetic testing is currently expensive and is not covered by all insurance plans, thus potentially leading to significant out of pocket expenses for patients, their families, and institutions. Because of the complexity of genetic testing, many tests require lengthy consent forms that take time and expertise 1507 Genetic Testing in Pediatric Epilepsy Figure 1. Classification of genetic variants. Variants are either benign or pathogenic, but determining into which category a previously undescribed variant falls can be complicated. Variants that do not change protein structure generally would be predicted to be benign. Variants that likely result in a change in protein structure is usually predicted to be pathogenic (for example, a premature stop codon or substitution of a hydrophobic amino acid in a transmembrane domain of a protein for a hydrophilic amino acid). Prediction modeling also considers conservation of specific amino acid sequences across species. A highly conserved residue (i.e., the same in multiple levels of organisms) is presumed to be important for protein function so that a variant changing a highly conserved amino acid is more likely to be pathogenic. Parental sequencing is also a useful tool in determining the significance of a VUS. If an affected child carries a de novo mutation in a gene that can be related to the patient’s phenotype, the variant is more likely to be pathologic than if an unaffected parent carried the same variant. Sometimes prediction programs offer conflicting conclusions and they can have suboptimal sensitivity and specificity in their prediction for the impact of a variant,80 leaving the final interpretation of the significance of a result up to the clinician. Epilepsia ILAE to properly complete and can be cumbersome for both families and providers. The wide availability of large-scale genetic testing brings an overlap between clinical medicine and research since research findings can be applied for clinical purposes. Foregoing the need for an a priori genetic diagnosis, broad genetic screening such as with WES allows gene discovery. A VUS identified in one patient can be reclassified as disease causing if similar associations are found in other patients. Clinical discovery of variants can inform basic research regarding mechanisms that provide therapeutic targets.5 In addition, simple clinical observation regarding therapeutic responses in specific conditions can lead to further discovery of involved mechanisms, such as was noted in the exacerbating effect of sodium channel blockers in Dravet syndrome.6 Despite the practical and theoretical challenges of genetic testing, genetic testing may offer advantages over retaining a diagnosis of “idiopathic” epilepsy. Certain results may guide and alter treatment decisions as in Dravet syndrome. The benefit of providing an answer to the diagnostic odyssey cannot be understated. Family anxiety can be eased, a more specific prognosis may be available, additional at-risk relatives can be tested, and a specific diagnosis may lead to networking with other similarly affected families. A diagnosis and its inheritance pattern may assist in future family planning; some families learn that they have a 25% or 50% risk of having another affected child, whereas other families are relieved to find out that their affected child carries a de novo mutation. Selected Genetic Epilepsy Syndromes Benign familial neonatal convulsions (BFNC) Benign familial neonatal convulsions (BFNC) is an autosomal dominant condition presenting with focal or generalized seizures, sometimes with apneas or autonomic symptoms, that generally begin between 2 and 8 days of life. Seizures resolve in approximately 67% of patients by 6 weeks of life. Neurodevelopment is usually normal, although there is an increased risk of subsequent epilepsy (16%).7 Those patients who do develop epilepsy often develop seizures suggestive of benign epilepsy with centrotemporal spikes (BECTS). Treatment with phenobarbital is usually initiated due to the flurry with which seizures present, with up to 30 per day; however, treatment may not affect seizure recurrence or cessation. Genes involved in BFNC include KCNQ3 (Kv7.3)8 and KCNQ2 (Kv7.2).9 Seventy percent of cases have a mutation identified in one of these genes, with KCNQ2 responsible for 90% of genetically identified cases.10 De novo and inherited KCNQ2 mutations have similar prognosis.11 Mutations in KCNQ2 and KCNQ3 have also been reported in patients with BECTS with and without a history of neonatal convulsions.12 KCNQ2 encephalopathy Some patients with KCNQ2 mutations experience a much more severe course, with epileptic encephalopathy, refractory seizures, and/or intellectual disability.13 In the first week of life, patients present with tonic seizures and an initial burst suppression electroencephalography (EEG) pattern. Seizures are typically eventually controlled with sodium channel blockers, but all patients have severe intellectual disability. Such outcomes have been reported to arise from de novo mutations and from families with a history of typical BFNC.14 Benign familial infantile convulsions (BFIC) Benign familial infantile convulsions demonstrate more genetic heterogeneity than BFNC. Onset is typically between 3 and 10 months and consists of brief seizures with Epilepsia, 56(10):1505–1514, 2015 doi: 10.1111/epi.13122 1508 M. A. Ream and A. D. Patel head and eye deviation, tonic stiffening, and cyanosis with unilateral clonic limb movements that become bilateral. Seizures are associated with occipitoparietal discharges that generalize.15 Seizures occur in clusters over 1–4 days and then generally subside within 1 year. Interictal EEG and development are normal. Cases with and without family history have a similar clinical course.16 The majority (70%) of cases of BFIC are linked to PRRT2 mutations,17 which are associated with pure BFIC, BFIC with choreoathetosis,18 and paroxysmal kinesigenic dyskinesia. Seizures generally respond well to antiepileptic medications including carbamazepine, phenobarbital, valproate, or zonisamide.19 However, if sodium channel blockers are used without effect, mutation in SCN2A must be considered.20 X-linked infantile spasms (CDKL5 and ARX) Mutations in CDKL5 are inherited in an X-linked dominant fashion and are a common cause of infantile spasms (IS) and early onset seizures. CDKL5 is responsible for 8% of early onset seizures (<9 months) in girls and 28% of early onset seizures with infantile spasms in girls.21 Frequency in boys is more difficult to ascertain due to fetal loss, but CDKL5 mutations were found in 3% of boys undergoing CDKL5 sequencing as part of an evaluation for early onset intractable epilepsy.22 CDKL5 was first described in cases of atypical Rett syndrome in patients with acquired microcephaly, developmental delay, limited language, hand apraxia, and chorea, but who had infantile onset epilepsy and severe hypotonia, and often lacked a period of regression.23 The first family described illustrates the phenotypic variability24; one sister had atypical Rett syndrome. Her twin sister had autism and mild-to-moderate intellectual disability without seizures. Their brother, who also carried the mutation, had LennoxGastaut syndrome, profound intellectual impairment, spastic quadriparesis, and cortical blindness. He eventually became “unresponsive” to environmental stimuli. Diagnosis of CDKL5 mutation may help prognosis and limit further testing but does not necessarily affect treatment. ARX mutation is a rare cause of X-linked recessive intellectual disability,25 either as a sole symptom or in combination with a variety of other neurologic abnormalities including infantile spasms, epilepsy, dystonia (Partington syndrome) and autism.26 Loss of function mutations are associated with brain malformations including lissencephaly (often with abnormal testes), hydranencephaly, and agenesis of the corpus callosum.27 ARX de novo mutations were found in 3 of 264 patients with epileptic encephalopathy (IS or Lennox-Gastaut syndrome),28 whereas mutation was found in 2.8% of boys undergoing ARX sequencing as part of an evaluation for early onset intractable epilepsy and 3% of boys with X-linked intellectual disability who were FRX negative.29 Seizure types associated with ARX mutations include IS, generalized tonic–clonic, myoclonic, absence seizures, and Epilepsia, 56(10):1505–1514, 2015 doi: 10.1111/epi.13122 complex partial. Microcephaly, macrocephaly, and increased and decreased tone have all been associated with mutations in boys. Intellectual impairment can be mild to severe, with more severe forms often associated with IS.30 Most reported cases are male, but female patients with ARX mutations can have learning and cognitive disabilities, autism, neuropsychiatric disorders (anxiety, depression, and schizophrenia), epilepsy, and agenesis of the corpus callosum. The severe phenotype seen in boys with intellectual impairment and IS can also be seen in girls if there is preferential X inactivation of the normal allele.31 ARX is considered an interneuronopathy resulting in loss of c-aminobutyric acid (GABA)ergic interneurons.32 This observation may eventually help in selecting specific antiepileptic drugs, for example, vigabatrin, which enhances GABA signaling, but studies on pharmacogenetics are still lacking. Glucose 1 transporter deficiency Glucose transporter deficiency syndrome type 1 (GLUTDS) is an autosomal dominant condition due to mutation in the glucose transporter and presents with seizures in the first year of life with acquired microcephaly, spasticity, and ataxia. Seizure types in infants include atypical absences and infantile spasms, and in older children generalized tonic–clonic seizures and typical absence.33 Other less common phenotypes include ataxia, exercise-induced dyskinesia, intellectual disability without seizures, and alternating hemiplegia of childhood.34 GLUT-DS has been associated with 10% of patients with early onset absence epilepsy and 1% of idiopathic generalized epilepsy. 35 In the classical phenotype, diagnosis can be made based on low cerebrospinal fluid (CSF) glucose content (<2.5 mmol/l) and low CSF to serum glucose ratio (generally <0.5)36; however, the sensitivity of CSF analysis has been questioned.37 Diagnosis can be confirmed by sequencing the SLC2A gene. Treatment with the ketogenic diet improves seizures and movement disorders. Steroids and carbonic anhydrase inhibitors can also be helpful.38 Pyridoxine-dependent epilepsy Pyridoxine-dependent epilepsy is caused by recessive mutations in ALDH1A7, the gene coding antiquitin, which is an a-amino adipic semialdehyde (AASA) dehydrogenase that is part of the lysine catabolism pathway. Deficiency will result in defective conversion of glutamate to GABA, leading to excess neuroexcitation.39 The abnormal metabolism can also cause structural brain defects that are not corrected by postnatal B6 supplementation.40 The classic form causes medication-resistant seizures in the first few hours of life,41 although atypical presentations have been described with later onset seizures and seizure-free periods without pyridoxine supplementation. Diagnosis can be suggested by elevated AASA or pipecolic acid in urine, plasma, and CSF whereas urine organic acid profile shows elevated lysine. A 1509 Genetic Testing in Pediatric Epilepsy lysine-restricted diet may be helpful in preventing the toxicity of accumulated metabolites and reduce seizure frequency.42 The most satisfying diagnostic method is observation of clinical and electrographic improvement within minutes of administering parenteral pyridoxine,43 although the short-term response is not always diagnostic. Definitive diagnosis is through CSF measurement and gene sequencing. such as carbamazepine and phenytoin has been associated with seizure aggravation and is contraindicated. A de novo genetic variation in the SCN1A gene occurs in approximately 80% of patients, mostly due to single nucleotide mutations or small insertions or deletions.52 Other genes recently associated with the Dravet phenotype include SCN2A, GABRG2, GABRA1, PCDH19, STXBP1, and HCN1.53,54 Pyridoxal phosphate–dependent epilepsy Mutation in PNPO, which encodes pyridoxine 50 -phosphate (P5P) oxidase, prevents conversion of pyridoxine to its biologically active form and results in P5P-dependent epilepsy. Seizures can start in utero, within hours of birth, or as late as 2 years of age.44 Seizures may present with a variety of characteristics including myoclonic, tonic, clonic, tonic status epilepticus, neonatal generalized tonic–clonic seizures, and electrical status epilepticus of sleep (ESES).44 Biochemical findings include elevated glycine and threonine on the plasma amino acid profile, elevated CSF L-DOPA and 3-methoxytyrosine, and reduced urine homovanillic acid and 5-hydroxyindoleacetic acid.45 Treatment with pyridoxal-5-phosphate 30–50 mg/kg/day divided in three to four doses can lead to reduction in seizure recurrence and improvement of cognitive functions.46 Generalized epilepsy febrile seizures plus (GEFS+) GEFS+ refers to an autosomal dominant epilepsy syndrome with incomplete penetrance. Patients present with generalized tonic–clonic seizures often associated with fever, usually presenting or continuing beyond 6 years of age when simple febrile seizures abate. They may also present with afebrile epileptic seizures. Other seizure types can be present as well. GEFS+ is associated with a variation in the SCN1A gene, which encodes the a1 subunit of a voltage gated sodium channel.55 Other genes associated with GEFS+ include SCN2A, SCN1B, GABA-A, GABRD, and GABRG2.56 Testing for these patients may not be helpful as a full-treatment response, and remission often occurs by the preteen years.57 Cerebral folate deficiency Cerebral folate deficiency is due to either autoimmunity against or mutation in FOLR and can be detected as low CSF methyl tetrahydrofolate (MTHF). Folate is converted to 5MTHF, which is used for production of purines and recycling of homocysteine into s-adenosylmethionine. Autoimmune cerebral folate deficiency presents as sleep disturbance and irritability in infancy and can progress to seizures, autism, dyskinesia, ataxia, and spasticity if untreated.47 Folate deficiency caused by receptor gene mutation presents in childhood as neurodegeneration with seizures, developmental delay, ataxia, hypotonia, and, in adolescents, severe polyneuropathy. Low MTHF can also be seen in 3-phosphoglycerate dehydrogenase deficiency (a congenital serine biosynthesis disorder), and mitochondrial disorders such as Kearns-Sayre syndrome or Alpers disease.48 Treatment of cerebral folate deficiency, either due to autoimmune or receptor mutation, is with folinic acid (0.5– 1 mg/kg/day).49 Folic acid is contraindicated for these patients, as folic acid competes at FR1 with the biologically active form MTHF and can worsen symptoms.50 Dravet syndrome (DS) Children with DS often present with generalized febrile or hemiconvulsive seizures that start during the first year of life.51 Eventually, myoclonic and other seizure types occur. As the seizures continue and often become treatment resistant, developmental slowing or regression occurs. Treatment with sodium channel blocking anticonvulsants Epilepsy limited to females with mental retardation (EFMR) Girls with epilepsy limited to females with mental retardation (EFMR) present in infancy with focal or generalized tonic–clonic febrile seizures, with clustering occurring within a few days of the first seizure. Often, these seizures are difficult to control with treatment. At seizure onset, developmental plateauing or regression occurs.58 Many of the girls have autistic features. A variation in PCDH19 encoding protocadherin 19 is felt to be associated with EFMR, with a unique mode of X-linked inheritance in which only heterozygous females and mosaic males are affected.59 Overlap with symptoms seen in Dravet syndrome can complicate the diagnosis. Therefore, PCDH19 testing should be considered in female patients with a Dravet-like phenotype or in those females with previous negative SCN1A testing. Progressive myoclonic epilepsy (PME) Adolescent-onset myoclonic epilepsy most often represents juvenile myoclonic epilepsy, but a small subset of these patients will later prove to have PME-targeted sequencing of EMP2A/laforin and EMP2B/malin (which cause Lafora progressive myoclonic epilepsy), and cystatin B (Unverricht-Lundborg disease) can distinguish these catastrophic diseases from juvenile myoclonic epilepsy in the early stages in which they may not be clinically distinguishable.60 A diagnosis of Lafora disease may lead to targeted therapy with a premature stop codon read-through drug (such as gentamicin) and avoidance of drugs that can exacerbate myoclonia and dementia such as sodium channel Epilepsia, 56(10):1505–1514, 2015 doi: 10.1111/epi.13122 1510 M. A. Ream and A. D. Patel blockers and GABAergic drugs.61 Such testing can be low yield and expensive and should be reserved for cases that are resistant to treatment. There are also panels with genes related to myoclonic epilepsy. A patient’s insurance coverage may help determine if a panel or multiple targeted genes done in series is more cost-effective. Genetic focal epilepsies There has been significant advancement recently in the genetics of focal epilepsies.62 Variation in several acetylcholine receptors led to autosomal dominant familial focal seizures, including nocturnal frontal lobe epilepsy and temporal lobe epilepsy.63,64 Focal epilepsy due to brain structural abnormalities also often has a genetic etiology as in DEPDC5.65 There is wide phenotypic variation in genes associated with focal epilepsies, as seen in KCNT1 and GRIN2A, both of which can cause focal epilepsy and epileptic encephalopathy.63,66,67 Such variability makes broad genetic evaluation much more practical than single gene sequencing in most cases. seizures.69 As new genes and associations are discovered, the indications for genetic testing may change in the future. For drug-resistant epilepsy, unless a specific disorder is highly suspected in the situations discussed in subsequent text, we recommend the diagnostic approach described in Figure 2. Initial biochemical screening should be done, when indicated, to uncover treatable metabolic disorders quickly while awaiting genetic confirmation. Simultaneously, genetic evaluation can be initiated with microarray followed by gene panel and then WES if diagnosis is still sought. Direct biochemical testing Seizures can be the presenting symptom of inborn errors of metabolism (IEMs) or can occur later in the course of dis- Epileptic encephalopathies Many of the above-listed genes and many additional genes (for example SCN8A, GNAO1, CHD2, SIK1, SYNGAP1, SLC6A1, KCNA2, and DNM1) can result in epileptic encephalopathy. Epilepsy panels cover the better-described genes. However, it is clear from exome-sequencing studies that many genes are potential candidates and many of these candidates fall within the same functional network.28,68 Computational biology will help us further delineate genes and gene networks involved in etiology and treatment of epilepsy as science advances. Proposed Algorithm for Diagnosis of Drug-Resistant Epilepsy If a genetic disorder is suspected as the cause of epilepsy, based on drug resistance, family history, or clinical phenotype, timely diagnosis may reduce overall cost, limit the “diagnostic odyssey” that can bring much anxiety to families, improve prognostication, and lead to targeted therapy. Recent ILAE recommendations indicate that genetic counseling should be available to all epilepsy patients, but that genetic evaluation should be undertaken at a tertiary level of epilepsy care, to which infants with seizure and patients of any age after failure of one antiepileptic drug should be referred.69 Generally, genetic testing is not recommended in drug-responsive epilepsy or at epilepsy onset, although comparative genomic hybridization (CGH) can be used as firsttier evaluation of patients with global developmental delay, which is a population that is at higher risk of epilepsy. Metabolic testing should be undertaken at the onset of epilepsy in infants without structural or syndromic cause of their Epilepsia, 56(10):1505–1514, 2015 doi: 10.1111/epi.13122 Figure 2. Algorithm for the diagnostic evaluation of drug resistant epilepsy. Epilepsia ILAE 1511 Genetic Testing in Pediatric Epilepsy ease. IEMs should be suspected as a potential cause of epilepsy in infants, particularly if the clinical history is not consistent with hypoxic ischemic encephalopathy or if there were in utero seizures. Other clinical scenarios for which to consider IEM include cases of myoclonic epilepsy, infantile spasms, atypical absences, epilepsia partialis continua, episodic decompensation, and when hypsarrhythmia or burst suppression is present on EEG (Table 2). In addition, by the time a patient has established drug-resistant epilepsy, they should have already undergone magnetic resonance imaging (MRI) to rule out structural lesions as the cause of epilepsy; such imaging may provide clues to IEMs that may coexist with brain lesions.70 All patients with suspected IEM should have initial metabolic screening (Table 3). Additional studies may be performed depending on the clinical scenario. Several authors have provided reviews to direct such testing in specific circumstances.71 Results can be obtained from metabolic evaluation much faster than from either targeted genetic studies or panels that include genes associated with these disorders, allowing for rapid initiation of specific treatments, when available. Genetic confirmation is helpful when there is a biochemical diagnosis and allows noninvasive screening of other family members if needed. Single gene sequencing Earlier we discussed a list of genetic epilepsies, but most have a variable phenotype, making identification of “classical phenotypes” less likely. However, there are a few condi- Table 2. Clinical situations in which to suspect an inborn error of metabolism. Neonates with history of in utero seizures Myoclonic epilepsy in infants Infantile spasms Atypical absence Epilepsia partialis continua Episodic decompensation Hypsarrhythmia Burst suppression MRI with metabolic pattern Table 3. Recommended initial metabolic screening Electrolytes with glucose complete blood count (CBC) Hepatic enzymes Plasma amino acids Urine organic acids Ammonia Lactate Acylcarnitine profile Consider cerebrospinal fluid (CSF) analysis If hypotonic: creatine phosphokinase (CK) tions in which targeted sequencing may be prudent due to issues related to treatment or prognosis and these include the following: Dravet syndrome, paternal transmission of early onset epilepsy and developmental delay in females, early onset absence epilepsy, and in some situations regarding pharmacologic management (Table 4). There are several online resources for identifying classical phenotypes and determining which commercial labs perform specific gene sequencing including GeneReviews (http://www.ncbi. nlm.nih.gov/books/NBK1116/) and genetests.org. Deprez et al. suggested an algorithm for targeted genetic evaluation of epilepsy occurring in the first year of life, but given the interval development of gene panels, his proposed approach that often requires sequencing of more than one gene for a given phenotype would be more costly than a gene panel, except perhaps in the conditions listed in Table 4. Chromosomal analysis Drug-resistant epilepsy, especially when associated with developmental delay or congenital anomalies, should prompt comparative genomic hybridization analysis (chromosomal microarray), which detects CNV. Balanced rearrangements and point mutations will not be detected, thus, limiting utility for diseases caused by single genes.72 CGH in patients with epilepsy and developmental delay has a 23.5% rate of abnormal findings.73 Yield increases when multiple abnormalities are present. Costs to obtain this testing have decreased in the past several years with many, but not all, insurance carriers providing reimbursement. Reflex to other forms of genetic testing, such as gene panels, when results are negative for CGH are available through some commercial labs. Karyotype can detect aneuploidy and balanced translocations not detectable by CGH with a resolution down to 3 Mb. A yield of 14% in epilepsy has been reported, although numbers were small.2 Several chromosomal disorders are associated with epilepsy, warranting karyotype if microarray is negative, and large genomic abnormality is expected based on phenotype. Gene panels Most cases of drug-resistant epilepsy are not so straight forward as to indicate a clear monogenetic diagnosis. Given variable expressivity, incomplete penetrance, multigeneic interactions, and de novo mutations lacking an informative family history, targeted single gene sequencing is not practical in most cases. In addition, the ILAE reported multiple genes for which a genetic diagnosis is considered very useful.74 If such diagnoses are not to be missed, casting a broad net with genetic testing can be justified. Utilizing NGS, panels were developed to evaluate genetic variation within multiple disease-related genes simultaneously. The quality of the testing relies on the coverage available at each base-pair position on the genes being tested.75 This becomes important, as depth of coverage varies Epilepsia, 56(10):1505–1514, 2015 doi: 10.1111/epi.13122 1512 M. A. Ream and A. D. Patel Table 4. Clinical situations that may warrant targeted gene sequencing Targeted gene sequencing SCN1A PCDH19 SLC2A1 POLG HLA-B*1502 P450 and other liver enzymes Clinical condition Advantage of testing Dravet syndrome. Consider testing if recurrent episodes of febrile status epilepticus, intractable tonic–clonic seizures during the first year of life, epileptic encephalopathy attributed to vaccination, and in adults with a history consistent with Dravet syndrome Females presenting with multiple clusters of brief febrile seizures and developmental delay or regression, particularly if there is a family history consistent with paternal transmission. Onset of absence seizures <4 years old, particularly if there is a family history of paroxysmal exercise-induced dyskinesia Prior to starting valproic acid in patients with drug resistant seizures and developmental delay or regression Prior to starting carbamazepine, oxcarbazepine, phenytoin and lamotrigine in patients of Asian descent Unexpected antiepileptic drug toxicity or unexpectedly high/low drug levels. (still under research, and not broadly recommended) between commercial labs, which employ different quality measures. Coverage of the genes may be equal to or greater than what is performed via WES. The number of genes available on a panel range from 20 to >400, depending on which company’s testing is utilized. Panels broadly related to a patient’s phenotype (e.g., myoclonic epilepsy, infantile onset epilepsy) may be the most cost–effective approach to genetic diagnosis because it limits the search to known epilepsy genes but covers multiple genes at once. When a panel of 265 genes was applied to 33 patients with refractory epilepsy, 16 received a specific diagnosis.75 Multiple gene panels of various sizes are available commercially. Companies tout their utility, with up to 22% sensitivity of one panel with 51 genes related to infantile onset epilepsy (GeneDx.com). Whole exome sequencing Currently the broadest net one could cast with clinical genetic evaluation is with WES because it allows simultaneous interrogation of all genes without limitation to known epilepsy genes and includes all exons in the genome.76 The American College of Medical Genetics recommends that exome sequencing be considered when there is a suspected genetic disease without a specific test available, in disorders with genetic heterogeneity that would require sequencing of multiple genes simultaneously for proper diagnosis, when there is a suspected genetic disorder but specific genetic tests are negative, and in some cases for prenatal testing (Table 5).77 WES has proven to be helpful in the diagnosis Table 5. Indications for WES Suspected genetic disease without a specific test available Disorders with genetic heterogeneity that would require sequencing of multiple genes simultaneously Genetic disorder is suspected, but specific genetic tests are negative Special prenatal circumstances Epilepsia, 56(10):1505–1514, 2015 doi: 10.1111/epi.13122 Avoidance of sodium channel blockers, aggressive seizure management, justification of stiripentol, bromides, etc. Prognosis and potential forthcoming treatment options Initiation of ketogenic diet Avoidance of potentially fatal liver failure starting as early as 2 months after initiation of valproic acid therapy Avoidance of potentially fatal reaction (Stevens-Johnson syndrome/toxic epidermal necrolysis) Avoidance of drugs metabolized through an aberrant P450 system of epilepsy. Protein-altering mutations in genes related to, or potentially related to, epilepsy were found in 9 of 10 patients with epileptic encephalopathy undergoing WES.68 Of 264 patients with epileptic encephalopathies, 329 de novo mutations were found, whereas only a fraction of these were known to be pathogenic. Such studies highlight the complexity of interpreting WES data and the potential for gene discovery if multiple patients with similar phenotypes carry similar variants or if variants occur in a common biochemical pathway.28 Commercially, 39% sensitivity for WES applied to epilepsy trios has been advertised (GeneDx.com). Definitive diagnostic yield has been reported as high as 45% in patients with neurodevelopmental disabilities, and these authors argue that WES followed by whole genome sequencing can be cost-effective and even less expensive than traditional molecular diagnostic approaches.78 In common application, WES results may vary; one retrospective study reported 14.3% diagnosis based on WES in treatment-resistant pediatric epilepsy,2 and another reported 27% yield for patients with a variety of neurodevelopmental conditions, including epilepsy.79 For the most useful interpretation of a patient’s WES, it is preferable to also obtain testing from both biologic parents to allow identification of de novo variants, which are more likely to be disease causing than variants inherited from unaffected parents. Although WES gives the largest volume of data regarding a patient’s genetics and requires no prior diagnosis, it has several limitations—including cost, turnaround-time, ethical concerns, and the potential for irrelevant or unwanted data—and should not be considered a first-line test. Reanalysis of data is sometimes also necessary. As more data are reported in the literature, some variants previously classified as of unknown significance may be reclassified as pathogenic. Thus, continuing to publish novel findings in the medical literature continues to be important and considering periodic repeat analysis of previously reported VUS may be beneficial. 1513 Genetic Testing in Pediatric Epilepsy Costs for WES range from $5,000 to >$14,000, with most insurance carriers not providing reimbursement as they consider such testing experimental, although institutional and bulk discounts may sometimes apply. Precertification is recommended and often requires multiple steps of appeals to obtain authorization. If metabolic testing, microarray, and gene panel are not informative but a genetic diagnosis is still sought, the physician and family must weigh the many pros and cons of pursing further genetic testing with WES. Summary The use of genetic testing in pediatric epilepsy is complicated and the list of known epilepsy genes changes almost daily. Also as reimbursement for testing changes, the most cost-effective approach may change over time. Currently we recommend reserving WES for the most elusive cases, but in time sending one genetic test with the broadest genetic coverage may become practical; however, there are still technical limitations that will need to be addressed as WES becomes more utilized. It would be ideal if WES could be tailored for epilepsy patients by optimizing coverage of known epilepsy genes. If this were to occur, further need for testing with a panel first would not be necessary and would ultimately lead to cost savings by reducing the total number of tests ordered. Alternatively, reflex testing to WES is an option when an epilepsy gene panel is negative. A few commercial companies currently offer this option. Careful consideration of each available test with its limitations must precede selection and ordering. A close partnership with genetics and genetic counseling is strongly recommend before pursuing testing that may reveal unexpected, and sometimes unrequested, results. Interpretation of complicated results should be performed only in collaboration with genetics providers unless the ordering neurologist has a strong background in and understanding of genetics. Genetic testing can play an important role in the care provided to patients with epilepsy. Disclosures Neither of the authors has any conflicts of interest to disclose that are relevant to this manuscript. 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