- Email: [email protected]
Diagnostic Yield of Epilepsy Panels in Children With Medication-Refractory Epilepsy
Accepted Manuscript Diagnostic Yield of Epilepsy Panels in Children with Medication-Refractory Epilepsy Eric Segal, M.D., Helio Pedro, M.D., Karen Valdez-Gonzalez, M.S., Sarah Parisotto, M.S., Felicia Gliksman, M.D., Stephen Thompson, M.D., Jomard Sabri, Evan Fertig, M.D. PII:
S0887-8994(15)30249-6
DOI:
10.1016/j.pediatrneurol.2016.06.019
Reference:
PNU 8940
To appear in:
Pediatric Neurology
Received Date: 16 October 2015 Revised Date:
22 June 2016
Accepted Date: 23 June 2016
Please cite this article as: Segal E, Pedro H, Valdez-Gonzalez K, Parisotto S, Gliksman F, Thompson S, Sabri J, Fertig E, Diagnostic Yield of Epilepsy Panels in Children with Medication-Refractory Epilepsy, Pediatric Neurology (2016), doi: 10.1016/j.pediatrneurol.2016.06.019. 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.
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Dated: June 21, 2016
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Dear Dr. Roach:
Thank you for your review of our study, “Diagnostic Yield of Epilepsy Panels in Children with
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Medication-Refractory Epilepsy.” We have found the reviewers’ comments very constructive and appreciate how their suggestions have strengthened the paper. The results, discussion, and
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tables have undergone major revisions to include more clinical information regarding the subjects with clinically significant results. We believe these changes further demonstrate that NGS can be used to identify mutations in patients with atypical presentations of well-defined syndromes.
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As requested, we have provided that genes tested in each panel. We agree that removing the subject with non-pathogenic abnormal CMA results provides a more homogenous sample. All statistics and tables have been revised accordingly. The cost of tests was inconsistent as the
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price points were different depending if the patient had Medicaid, private payer insurance, charity care, or paid directly to the testing company.
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Sincerely,
Eric BJ Ségal, MD Northeast Regional Epilepsy Group 20 Prospect Avenue Suite 801 Hackensack, NJ 07601 Phone: 201-343-6676 Fax: 201-343-6689 Email: [email protected]
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Title: Diagnostic Yield of Epilepsy Panels in Children with Medication-Refractory Epilepsy
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Eric Segal, M.D.1, Helio Pedro, M.D.2, Karen Valdez-Gonzalez, M.S.2, Sarah Parisotto M.S.2,
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Felicia Gliksman, M.D.3, Stephen Thompson, M.D.3, Jomard Sabri1, Evan Fertig, M.D.1
Northeast Regional Epilepsy Group, 20 Prospect Avenue, Hackensack, NJ 07601
2
Hackensack University Medical Center, Division of Genetics, Department of Pediatrics, 30
Prospect Avenue, Hackensack NJ 07601 3
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Hackensack University Medical Center, Division of Pediatric Neurology, Department of
Title character count: 74
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Pediatrics, 30 Prospect Avenue, Hackensack NJ 07601
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Running title character count: 44 Abstract word count: 253 Main text word count: 1908 Number of tables: 4 Number of figures: 0 References: 12
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Running title: Next-Generation Sequencing in pediatric epilepsy Key words: gene panel, next-generation sequencing, children, epilepsy Correspondence: Eric BJ Ségal, MD Northeast Regional Epilepsy Group 20 Prospect Avenue Suite 801 Hackensack, NJ 07601 Phone: 201-343-6676 Fax: 201-343-6689 Email: [email protected]
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Abstract
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Introduction: When there are no chromosomal variations found, patients with suspected genetic etiologies can be tested using next-generation sequencing(NGS), known as epilepsy panels.
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Objective: The primary objective of this study was to examine the diagnostic yield of nextgeneration sequencing(NGS) seizure panels, known as epilepsy panels, in medication-resistant epilepsy subjects between 0-18 years old in subjects with non clinically-significant comparative genomic hybridization microarray (array-CGH) results. Methods: This is a single-center retrospective chart review of the diagnostic-yield of nextgeneration sequencing(NGS) seizure panels, known as epilepsy panels, in medication-resistant epilepsy subjects between 0-18 years old in subjects with non clinically-significant comparative genomic hybridization microarray(microarray-CGH) results from January 2011 to December 2014. The primary endpoint was the yield of clinically-significant NGS results.
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Results: Forty-nine subjects (21 male) were identified as having medication-refractory epilepsy with non-clinically-significant array-CGH results. NGS abnormalities were seen in 28 subjects(57%): 7 of these subjects(25%) had clinically significant findings. Mutations were found in the SCN1A gene in 3 subjects, in the PCDH19 gene in 2 subjects, and in DLG3 and in MECP2, TSC2, and SLC9A6 genes 1 subject each. Only the MECP2 mutation was found to be pathogenic in this last subject. The additional yield of NGS with uninformative CMA was 14%. Positive findings were primarily seen in those with Dravet Syndrome, all with SCN1A mutations(42% of clinically significant results). Given the small sample size, a larger prospective study would be helpful to determine the clinical yield of NGS.
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Conclusion: NGS seizure panels could be a useful tool in the diagnosis of non-acquired pediatric medication-refractory epilepsy with uninformative array-CGH.
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Introduction
Forty-seven percent of patients with refractory epilepsy have cryptogenic etiologies.i In addition
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to a comprehensive understanding of a patient’s history, examination, electroencephalogram, brain magnetic resonance imaging, many of these patients will undergo genetic testing to further
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elucidate a possible etiology. Genetic testing may include metabolic evaluation, chromosomal analysis, and molecular testing targeting epilepsy.
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In many epilepsy centers, molecular microarray testing such as array comparative genomic hybridization (microarray-CGH) is one of the first-line diagnostic tests in order to understand epilepsy etiology. This diagnostic test can detect abnormalities such as microdeletions,
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microduplications, copy number variants, and regions of homozygosity.ii
When there are no chromosomal variations found, patients with suspected genetic etiologies can be tested using next-generation sequencing(NGS), known as epilepsy panels. NGS provides the ability to analyze multiple genes simultaneously. NGS technologies can more effectively identify monogenomic epilepsies. Different laboratories have established panels of genes that relate primarily to epilepsy.
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The costs of NGS can be prohibitively high and often denied for patients with government-
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subsided insurance. The practice in our Center is to reserve NGS for patients with non-acquired medication-refractory epilepsy. Given the cost and challenge of obtaining the test, it is important to determine the diagnostic yield of NGS in the pediatric epilepsy population. Mercimek-
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Maghmuoglu, et. al. recently studied the diagnostic yield of NGS testing of pediatric epileptic encephalopathy.iii They found a 45% yield of clinically significant NGS abnormalities with 4.5%
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of the subjects had metabolic disorders diagnosed via this molecular testing. Lemke, et al. developed an epilepsy-NGS tool testing 265 genes and tested a pilot population of 33 adults and children of different clinical phenotypes and found a diagnostic yield of 48%.iv The goal of this study is to evaluate the diagnostic yield of NGS in children with non-acquired medication-
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refractory epilepsy who already have non-clinically significant chromosome microarray,
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metabolic, and neuroimaging.
Methods
A retrospective chart review was performed searching for subjects between 0-18 years of age with medication-refractory epilepsy subjects who had genetic testing performed from 2011-2014. Subjects were considered to be medication refractory after poor response to at least two different
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neurologists at Hackensack University Medical Center.
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anti-convulsant medications.v Subjects were referred to the genetics department from the
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Subjects who did not have both chromosomal microarray-CGH and next-generation sequencing panels were excluded.
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Charts were reviewed for results of brain imaging studies, metabolic evaluation, chromosomal microarray-CGH analysis, single gene molecular analysis and NGS panels. Metabolic evaluation included plasma amino acids, urine organic acids and based on clinical phenotype other metabolic tests were performed. This could include urine acyclglycines, carnitine levels,
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and lumbar puncture for neurotransmitters.
The array-CGH were performed by a variety of vendors depending on patient insurance contracts or hospital agreement (Clairsure® 190K probe Quest Diagnostics, Nichols Institute, San Juan
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Capistrano, CA; Affymetrix® 170K probe Laboratory Corporation of America Holdings, Research Triangle Park, NC; Agilent® 180K probe Mayo Medical Laboratories, Rochester, MN;
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Oligonucleotide array Bioreference Laboratories, Elmwood Park, NJ).
Epilepsy NGS testing was performed at either GeneDx (Gaithersburg, MD, USA) or at Courtagen Life Sciences (Woburn, MA, USA). Specific genes sequenced are listed in Table 4. Laboratory selection was mostly based on insurance approval and hospital agreements.
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Demographic information, clinical data related to seizures and epilepsy, and test outcomes were
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extracted by a clinical research assistant and reviewed by board certified genetic counselors, medical geneticist and a pediatric epileptologist. Etiologies for epilepsy were categorized using
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the ILAE 2010 recommendations.vi
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Data included gender, age of onset of seizures, history of developmental delay, first degree family history of seizures or epilepsy, prevalence of dysmorphic features, abnormal neurological exam (other than developmental delay), seizure types and history of abnormal MRIs. Descriptive statistics were utilized to convey the results of the analysis and to quantify the diagnostic yield of
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Results
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each test.
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A total of 49 subjects were identified for this study(Table 1). Twenty-one of the subjects were male. The mean onset of epilepsy was 2.6 (SD±0.6) years old.
Thirty subjects (61%) demonstrated developmental delay. There was a family history of epilepsy in 15 subjects (31%). Ten subjects (20%) had non-neurological medical problems.
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Physical exam findings revealed 2 subjects (4%) had dysmorphic features and 18 subjects (36%)
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had abnormal neurological examinations. Neither of the subjects with dysmorphic features were
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found to have clinically significant NGS results.
Diagnostic testing showed 17 subjects (35%) had abnormal MRI findings. Most of these findings
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consisted of non-specific findings such as partial agenesis of corpus callosum. However, 2 subjects did have malformations of cortical development (i.e. pachygyria and polymicrogyria). Neither of those subjects had any clinically significant molecular testing. None of the subjects
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with clinically significant NGS had abnormal MRIs.
Using the 2010 ILAE recommendations for classification of etiologies, 14 subjects (29%) had presumed genetic, 1 patient (2%) had presumed metabolic, 5 subjects (10%) had structural, and
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29 subjects (59%) had unknown etiologies(Table2).
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Ten subjects (20%) had abnormal, non-clinically significant array-CGH results. NGS abnormalities were seen in 28 subjects (57%). Seven of these subjects (25%) had clinically significant findings (Table 3). Of these 7 subjects, 3 had SCN1A mutations, 2 had PCDH19 mutations, 1 had a DLG3 mutation, and 1 had mutations in the MECP2, TSC2, and SLC9A6 genes. For this final patient, it was determined that only the MECP2 mutation was pathogenic, while the other two variants were found to be non-significant.
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At the time of testing, none of the subjects with clinically-significant NGS results had
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dysmorphism, non-neurological problems, or clinically significant MRI findings.
Subject 1 was 3.5 months old at age of seizure onset. The patient’s seizures were initially focal afebrile seizures. This patient did go on to develop myoclonic seizures at 13 months of age and
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found to have febrile-triggered seizures. Developmental delay was not present. MRI brain
demonstrated minimal nonspecific periventicular white matter T2 intensity as well as subtle
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increased signal intensity of both hippocampi without volume loss.
Subject 2’s seizures began at 14 months of age without febrile seizures. However, the patient later had febrile-induced seizures. The patient had an inconsistent response to sodium-channel agents. For example, oxcarbazepine and lacosamide exacerbated the subject’s seizures and
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lamotrigine did not. The clinical history was significant for developmental delays in the gross motor, fine motor, and speech domains. On exam, the patient was found to have appendicular hypotonia. At a previous institution, prior to NGS testing, this subject underwent a surgical
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evaluation including subdural electrode placement without resection. MRI performed prior to
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surgical evaluation was concerning for a nonspecific linear T2/FLAIR hyperintense signal within the left anterior corona radiate which was deemed an incidental finding. Subject 3’s first seizure was a febrile seizure at 18 months of age. This patient underwent a language regression around the same time as onset of seizures. Like subject 2, the patient has heat-triggered seizures. The patient’s MRI demonstrated a nonspecific foci of T2 prolongation within the left frontal and right parietal subcortical white matter. This finding did not evolve on
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repeat imaging and was deemed an incidental finding. The subject’s father was found to have
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the same genetic mutation.
The positive MECP2 mutation was found in a female who started having multifocal seizures at 5 weeks of life. The patient met criteria for malignant migrating partial epilepsy in infancy. The
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patient underwent genetic testing at that time prior to developing her clinical manifestations including decelerated head growth, developmental delay, and non-purposeful use of her hands.
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Upon presentation, the subject was found to have a normal MRI brain. The subjects exam was significant for axial and appendicular hypotonia.
Subject 6 initially presented with epileptic spasms that evolved to drop seizures. The patient was found to have global developmental delays, axial and appendicular hypotonia, and Autistic
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Spectrum Disorder. The patient’s MRI was normal. Both subjects that tested positive for PCDH19 mutations were female with hypotonia and global
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developmental delay. Subject 4 was also diagnosed with intellectual disability and ADHD.
Discussion
In our population, NGS helped diagnose 14% of children with medication refractory epilepsy. NGS represents the next step in the genetic diagnostic pathway. Previous studies have studied NGS yield in either mixed-age population or children with epileptic encephalopathies. To our
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knowledge, this is the first report of the diagnostic yield of pediatric medication-refractory
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epilepsy who have non-clinically significant array-CGH.
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NGS testing yielded a genetic sequence variant in more than half of all subjects, and of these,
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14% had clinically significant mutations.
The majority of significant results were found in subjects with a SCN1A mutation. This mutation, identified in Dravet Syndrome patients, is beneficial for pharmacologic purposes. There are anti-convulsant medications that are contraindicated and others known to be more
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effective for this specific syndrome.vii Early knowledge of an SCN1A mutation, therefore, can save the patient from unnecessary treatments and hospitalizations. In our cohort, these genetic findings resulted in discontinuation of sodium blocking agents as well as further conversations
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with families regarding prognosis.
Many patients with SCN1A mutations have a phenotype consistent with Dravet Syndrome. This
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would include febrile seizure beginning during the first year of life typically beginning between 5-8 months of age. While some of the subjects did later develop more typical features consistent with Dravet Syndrome, the atypical age of onset of seizures, seizure types, and inconsistent response to sodium-channel agents warranted broader testing.viii Two subjects diagnosed with PCDH19 mutation has led to not only more informative counseling but also possible more targeted therapies. Patients with PCDH19 mutations can have clinical
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histories similar to patients with Dravet Syndrome.ix The genetic diagnosis can open-up further
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therapies.x For example, there are on-going studies using new-medications for this particular epilepsy etiology (clinical trial ID: NCT02358538).
The case of subject 7 highlights the diagnostic advantage of early use of NGS in children with
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medication-refractory epilepsy and previously negative testing. The positive MECP2 mutation was found prior to developing her clinical manifestations including decelerated head growth,
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developmental delay, and non-purposeful use of her hands. With this mutation, the patient also met criteria for the Hanefeld Variant of atypical Rett Syndrome.xi Additionally, to our knowledge, MECP2 mutations have not been described in patients with malignant migrating partial epilepsy of infancy. Although this particular mutation did not change epilepsy
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management, it did help with anticipatory guidance for the family.
Another patient had a positive DLG3 mutation which has been associated with cortical hyperexcitability in the NMDA pathway.xii At the time of diagnosis, there was no specific
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therapy available for mutations involving the NMDA receptor, however, it is conceivable that
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this patient may respond to anti-glutameteric agents such as perampanel. Consideration must be given to the limitations of the study. It is retrospective, based in a single center, and involves a small sample size. In addition, different vendors were used for NGS as well as for array-CGH testing which may lead to further inconsistencies. Larger, prospective studies on children with medication refractory epilepsy would be helpful to determine the clinical yield of NGS.
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This study demonstrates the yield of NGS testing represents the next step in diagnosing children
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with non-acquired medication-refractory epilepsy. This testing leads to more informed
conversations regarding prognosis, family planning, and in some instances more targeted
therapies. As more mechanism-specific agents become available, it is feasible that appropriate
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identification can lead to a more rational and personalized approach of epilepsy treatment.
Conclusion:
Our study demonstrates that NGS can be a clinically useful diagnostic test for establishing an
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etiology for medication-refractory epilepsy and improving accuracy of prognosis and clinical management. Clinicians should consider using NGS in the evaluation of medication refractory epilepsy patients in whom neuroimaging and typical standard genetic testing has not yielded
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clinically significant results.
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Table 1 – Clinical characteristics of all subjects(n=49) 21/28
Mean age of onset of seizures
2.6 (range 0-17 years old)
Developmental delay
30
Family history of seizures/epilepsy
15
Additional medical history
1
Dysmorphic features
2
Abnormal neurological exam
18
Abnormal MRI
17
Abnormal array-CGH
10 28
Clinically significant abnormal NGS
7
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Abnormal NGS
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Boys/Girls
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Total(%) 14 (29%) 1 (2%) 5 (10%) 29 (59%)
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Diagnosis Genetic Metabolic Structural Unknown
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Table 2 – Results of array-CGH and NGS grouped by epilepsy diagnosis Clinicallysignificant NGS(%) 6 (85.7%) 0 1 (14.2%) 0
Abnormal arrayCGH(%) 1 (10%) 1 (10%) 1 (10%) 7 (70%)
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Table 3 – Clinical characteristics Age of Testing (years) 4.5
Developmental MRI Delay Prior to Findings Seizures No Incidental
2
Yes
8
No
PCDH19 PCDH19
Age of Onset (years) Missense 0.25 (febrile) Frameshift 1.2 (afebrile) Missense 1.5 (febrile) Missense 0.6 Missense 0.8
SCN1A
2
SCN1A
3
SCN1A
4 5
13 2.5
Yes Yes
6 7
DLG3 MECP2
Missense Missense
4 0.2
0.6 0.1
Epilepsy Syndrome
Incidental Incidental
Yes Yes
Normal Normal Normal Normal
Febrile GTC, hemiconvulsive afebrile seizures, GTC Febrile seizures, focal dyscognitive Hemi-clonic GTC, myoclonic jerks Spasms Hemi-clonic, dyscognitive
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Table 4 Gene Panels Episeek® (Courtagen)
Seizure Semiology
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1
Type of Mutation
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Gene
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Subject
Genetic Mutations
KANSL1 CNTNAP2, MBD5, NRXN1, SLC9A6, TCF4, UBE3A CHRNA2, CHRNA4, CHRNB2, LGI1
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Koolen-de Vries Syndrome Angelman/Angelman-like/PittHopkins Autosomal Dominant Focal Epilepsies Benign familial infantile seizures Benign familial neonatal seizures Benign familial neonatal-infantile seizures Cerebral folate deficiency Creatine deficiency syndromes EAST/SeSAME syndrome Familial infantile myoclonic epilepsy Generalized epilepsy with febrile seizures Hyperekplexia Joubert Syndrome
Juvenile Myoclonic Epilepsy Migraine Neuronal Ceroid Lipofucsionses Peroxisomal Biogenesis Disorders
PRRT2 KCNQ2, KCNQ3 SCN2A
FOLR1 GAMT, GATM, SLC6A8 KCNJ10 TBC1D24 GARBG2, SCN1A, SCN1B, SCN2A ARHGEF9, GLRA1, GLRB, GPHN, SLC6A5 AHI1, ARL13B, CC2D2A, CEP290, INPP5E, NPHP1, OFD1, RPGRIP1L, TMEM216, TMEM67 BRD2, CACNB4, EFHC1, GABRA1 ATP1A2, BRAF, CACNA1A CLN3,CLN5,CLN6,CLN8,CTSD,CTSF,DNAJC5,KCTD7,MFSD8,PPT1, TPP1 PEX1,PEX2,PEX3,PEX5,PEX6,PEX7,PEX10,PEX12,PEX13,PEX14, PEX16, PEX19,PEX26
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Progressive Myoclonic Epilepsy Ras/MAPK pathway dysregulation Rett/atypical Rett Syndromes Brain or Nervous System Malformations
ASAH1,CSTB,EPM2A,FOLR1,GOSR2,KCTD7,NHLRC1,PRICKLE1, SCARB2 BRAF,HRAS,KRAS,MAP2K1,MAP2K2,NF1,NRAS,PTPN11,RAF1,SOS1, SPRED1
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CDKL5,FOXG1,MBD5,MECP2,MEF2C AHI1,ARFGEF2,ARL13B,ASPM,ATR,BRAF,BUB1B,C12orf57,CASK,CBL,CC2D2A,CC DC88C,CDK5RAP2,CDON,CENPJ,CEP152,CEP290, COL18A1, COL4A1, CPT2, DCLK2, DCX, ELP4, EMX2, EOMES, FGF8, FGFR3, FKRP, FKTN, FLNA, GLI2, GLI3,GPR56,KAT6B,LAMA2,LARGE, LIG4, MCPH1, MED17, MLC1, NHEJ1, NPHP1, OFD1, PAFAH1B1, PAX6, PCNT, PEX7, PIK3CA, PIK3R2, PLA2G6, PNKP, POMGNT1,POMT1, POMT2, QBP1, PTCH1, RAB3GAP1, RARS2, RELN, SERPINI1, SHH, SHOC2, SIX3, SLC25A19, SNAP25, SNAP29, SRPX2, STIL, STRADA, TGIF1, TSEN2,TSEN34, TSEN54,TUBA1A,TUBA8,TUBB2B,VANGL1,VRK1,WDR62, ZIC2 ALG1,ALG11,ALG12,ALG13,ALG2,ALG3,ALG6,ALG8,ALG9,B4GALT1,COG1,COG4, COG5,COG6,COG7,COG8,DDOST, DOLK,DPAGT1, DPM1, DPM3, MAGT1, MGAT2, MOGS, MPDU1, MPI, NGLY1, PGM1, PMM2, RFT1, SLC35A1, SLC35A2, SLC35C1, SRD5A3, TMEM165, TUSC3 AKT3,CNR1,CNR2,CYP2C19,CYP2C9,CYP3A4,CYP3A5,DAGLA,FAAH,GPR55,MGLL ,MTOR
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Congenital Disorder of Glycosylation
Drug Metabolism and Cannabinoid Receptors and Pathways Early onset Epileptic Encephalopathies or Infantile Spasms
ALDH7A1,ATN1,BRAT1,CACNA1A,CACNA1H,CACNB4,CASR,CHD2,CHRNA2,CHR NA4,CHRNB2,CLCN2,CNTN2,CSTB,DEPDC5,EFHC1, EFHC2, PM2A, GABRA, GABRB3, GABRD, GABRG2, GOSR2, GPR98, GRIN1, GRIN2A, GRIN2B, JRK, KCNMA1, KCNQ2, KCNQ3, KCTD7, MBD5, ME2, HLRC1, PCDH19, PRICKLE1, PRICKLE2, PRRT2, SCARB2, SCN10A, SCN11A, SCN1A, SCN1B, SCN2A, SCN3A, SCN3B, SCN4A, SCN4B, SCN5A, SCN7A, SCN8A, SCN9A, SLC2A1,TBC1D24
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Generalized/Myoclonic/Absence Epilepsies/Febrile Seizures
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GABA Receptors
ALDH7A1,ARHGEF9,ARX,ATP6AP2,CDKL5,CNTNAP2,FH,FOXG1,GABRG2,GRIN2 A,GRIN2B,HNRNPU,KCNQ2,KCNT1, LIAS, MAGI2, MEF2C, EDD4L, NRXN1, PCDH19, PLCB1, PNPO, POLG, PRRT2, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SCN9A, SLC25A22, SLC2A1, SLC6A4, SLC9A6, SPTAN1, ST3GAL3, STXBP1, TCF4, TSC1, TSC2, ZEB2 GABBR1,GABBR2,GABRA2,GABRA3,GABRA4,GABRA5,GABRA6,GABRB1, GABRB2,GABRE,GABRG1,GABRG3,GABRP,GABRQ,GABRR1, GABRR2,GABRR3
Mitochondrial Dysfunction
Protein and Carbohydrate Metabolism
Selected inborn errors of metabolism
Storage disease and organelle dysfunction including lysosomal
ADCK3, APTX, BCS1L, C12orf65, COQ2, COQ9, COX10, COX15, DLD, LRPPRC, NDUFA2, NDUFAF6, NDUFS1, NDUFS3, NDUFS4, NDUFS7, NDUFS8, NDUFV1, OPA1, PC, PDHA1, PDHX, PDSS1, PDSS2, POLG, SCO2, SDHA, SURF1, TACO1, VDAC1 ABCC8, ACY1, ADSL, AGA, ALDH4A1, ALDH5A1, ALDH7A1, AMT, ARG1, ATPAF2, BTD, CTSA, DPYD, ETFA, ETFB, ETFDH, FH, FOLR1, FUCA1, GCDH, GCSH, GLDC, GNE, HPD, HYAL1, L2HGDH, MOCS1, MOCS2, NEU1, PANK2, PGK1, PNPO, PRODH, QDPR, SLC17A5, SLC25A15 SLC46A1, SUMF1, SUOX ABAT, ACOX1, ATIC, ATP5A1, ATP7A, BCKDHA, BCKDHB, C12orf65, DBT, DDC, DHCR7, GLUD1, GLUL, HSD17B10, HSD17B4, KCNJ11, MGME1, MMACHC, MTHFR, MTR, MTRR, NDUFA1, PHGDH, PSAT1, SLC19A3, SUCLA2, TMEM70, VDAC1 ARSA, ARSB, ASPA, EIF2B1, EIF2B2, EIF2B3, EIF2B4, EIF2B5, FUCA1, GALC, GALNS, GFAP, GLB1, GNE, GNPTAB, GNPTG, GNS, GUSB, HEXA, HEXB, SNAT,
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IDS, IDUA, MCOLN1, MLC1, NAGLU, NOTCH3, NPC1, NPC2, PLP1, PSAP, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, SDHA, SGSH, SMPD1, SUMF1, TREX1
Syndromic disorders with epilepsy and others
ABCC2, ASPA, ATP1A2, ATP1A3, ATP2A2, BCKDK, CCL2, CLCNKA, CLCNKB, CNTNAP2, CPT1A, DYRK1A, FGD1, FLVCR2, FOXH1, GJD2, GUSB, HCN1, HCN2, HCN3, HCN4, HERC2, IDH2, KCNA1, KCNAB1, KCNJ1, KCNV2, KIAA1279, KMT2D, LBR, LGI1, MAPK10, NDE1, NIPBL, NODAL, RAI1, RBFOX1, NASEH2C, RTTN, SETBP1, SGCE, SLC1A3, SLC4A10, SMC1A, SMC3, SNIP1, ST3GAL5, TBX1, TRPM6, VPS13A, VPS13B, ZEB2
KANSL1 CNTNAP2, MBD5, NRXN1, SLC9A6, TCF4, UBE3A CHRNA2, CHRNA4, LGI1 PRRT2 KCNQ2, KCNQ3 SCN2A FOLR1 GAMT, GATM, KCNJ10 TBC1D24
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Koolen-de Vries Syndrome Angelman/Angelman-like/PittHopkins Autosomal Dominant Focal Epilepsies Benign familial infantile seizures Benign familial neonatal seizures Benign familial neonatal-infantile seizures Cerebral folate deficiency Creatine deficiency syndromes EAST/SeSAME syndrome Familial infantile myoclonic epilepsy Generalized epilepsy with febrile seizures Juvenile Myoclonic Epilepsy Neuronal Ceroid Lipofucsionses Progressive Myoclonic Epilepsy Ras/MAPK pathway dysregulation Rett/atypical Rett Syndromes Brain or Nervous System Malformations Early onset Epileptic Encephalopathies or Infantile Spasms
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GeneDx –Comprehensive Epilepsy Panel Epilepsy Syndrome Genetic Mutations
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storage disorders and leukodystrophies
GARBG2, SCN1A, SCN1B, SCN2A
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CACNB4, EFHC1, GABRA1 CLN3, CLN5, CLN6, CLN8, CTSD, DNAJC5, KCTD7, MFSD8, PPT1, TPP1 CSTB,EPM2A,FOLR1,GOSR2,KCTD7,NHLRC1,PRICKLE1, SCARB2 BRAF,HRAS,KRAS,MAP2K1,MAP2K2,NF1,NRAS,PTPN11,RAF1,SOS1, SPRED1
Generalized/Myoclonic/ Absence Epilepsies/Febrile Seizures
Generalized/Myoclonic/Absence
CDKL5,FOXG1,MBD5,MECP2,MEF2C PNKP, SLC25A19, SRPX2 ALDH7A1, ARX,ATP6AP2,CDKL5,CNTNAP2, FOXG1,GABRG2,GRIN2A, GRIN2B, KCNQ2, LIAS, MAGI2, MEF2C, NRXN1, PCDH19, PNPO, POLG, PRRT2, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SLC25A22, SLC2A1, SLC6A4, SLC9A6, SPTAN1, STXBP1, TCF4, TSC1, TSC2, ZEB2 ALDH7A1, CACNB4, CHRNA2, CHRNA4,CHRNB2,CSTB, GABRA, GABRB3, GABRG2, GOSR2, GRIN2A, GRIN2B, KCNQ2, KCNQ3, KCTD7, PRICKLE1, PRRT2, SCARB2, SCN1A, SCN1B, SCN2A, SLC2A1
ALDH7A1, CSTB, GABRD, GABRG2, GOSR2, GRIN1, GRIN2A, GRIN2B, KCNQ2,
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KCNQ3, KCTD7, MBD5, PRICKLE1, PRRT2, SCARB2, SCN1A, SCN1B, SCN8A, SLC2A1
Protein and Carbohydrate Metabolism
ADSL, ALDH7A1, FOLR1, PNPO, SLC25A15
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Epilepsies/Febrile Seizures
i
ix
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Kwan, Patrick, and Martin J. Brodie (2012). Early identification of refractory epilepsy New England Journal of Medicine 342.5 (2000): 314-319. ii Pong, A. W., Pal, D. K., & Chung, W. K. (2011). Developments in molecular genetic diagnostics: an update for the pediatric epilepsy specialist. Pediatric neurology, 44(5), 317-327. iii Lemke, J. R., Riesch, E., Scheurenbrand, et. al, (2012). Targeted next generation sequencing as a diagnostic tool in epileptic disorders. Epilepsia, 53(8), 1387-1398. iv Mercimek-Mahmutoglu, S., Patel, J., Cordeiro, D., et. Al. (2015). Diagnostic yield of genetic testing in epileptic encephalopathy in childhood. Epilepsia, 56(5), 707-716. v Kwan P, Arzimanoglou A, Berg AT, et. Al. (2010). Definition of drug resistant epilepsy: consensus proposal by the ad hoc Task Force of the ILAE Commission on Therapeutic Strategies. Epilepsia 51(6) 1069-77. vi Berg, A. T., Berkovic, S. F., Brodie, M. J., et. Al., (2010). Revised terminology and concepts for organization of seizures and epilepsies: report of the ILAE Commission on Classification and Terminology, 2005–2009. Epilepsia, 51(4), 676-685. vii Chiron, Catherine, and Olivier Dulac. "The pharmacologic treatment of Dravet syndrome." Epilepsia 52.s2 (2011): 72-75. viii Dravet, C (2011). The core Dravet syndrome phenotype. Epilepsia, 52(Suppl.2):3-9.
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Depienne, C., Bouteiller, D., Keren, B., et. Al., (2009). Sporadic infantile epileptic encephalopathy caused by mutations in PCDH19 resembles Dravet syndrome but mainly affects females.PLoS Genet, 5(2), e1000381. x Marini, C., Darra, F., Specchio, N., et. Al. (2012). Focal seizures with affective symptoms are a major feature of PCDH19 gene–related epilepsy. Epilepsia, 53(12), 2111-2119. xi Neul, J. L., Kaufmann, W. E., Glaze, et. Al. (2010). Rett syndrome: revised diagnostic criteria and nomenclature. Annals of neurology, 68(6), 944-949. xii Qu, M., Aronica, E., Boer, K., et. Al. (2009). DLG3/SAP102 protein expression in malformations of cortical development: a study of human epileptic cortex by tissue microarray. Epilepsy research, 84(1), 33-41.
S0887-8994(15)30249-6
DOI:
10.1016/j.pediatrneurol.2016.06.019
Reference:
PNU 8940
To appear in:
Pediatric Neurology
Received Date: 16 October 2015 Revised Date:
22 June 2016
Accepted Date: 23 June 2016
Please cite this article as: Segal E, Pedro H, Valdez-Gonzalez K, Parisotto S, Gliksman F, Thompson S, Sabri J, Fertig E, Diagnostic Yield of Epilepsy Panels in Children with Medication-Refractory Epilepsy, Pediatric Neurology (2016), doi: 10.1016/j.pediatrneurol.2016.06.019. 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.
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Dated: June 21, 2016
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Dear Dr. Roach:
Thank you for your review of our study, “Diagnostic Yield of Epilepsy Panels in Children with
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Medication-Refractory Epilepsy.” We have found the reviewers’ comments very constructive and appreciate how their suggestions have strengthened the paper. The results, discussion, and
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tables have undergone major revisions to include more clinical information regarding the subjects with clinically significant results. We believe these changes further demonstrate that NGS can be used to identify mutations in patients with atypical presentations of well-defined syndromes.
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As requested, we have provided that genes tested in each panel. We agree that removing the subject with non-pathogenic abnormal CMA results provides a more homogenous sample. All statistics and tables have been revised accordingly. The cost of tests was inconsistent as the
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price points were different depending if the patient had Medicaid, private payer insurance, charity care, or paid directly to the testing company.
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Sincerely,
Eric BJ Ségal, MD Northeast Regional Epilepsy Group 20 Prospect Avenue Suite 801 Hackensack, NJ 07601 Phone: 201-343-6676 Fax: 201-343-6689 Email: [email protected]
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Title: Diagnostic Yield of Epilepsy Panels in Children with Medication-Refractory Epilepsy
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Eric Segal, M.D.1, Helio Pedro, M.D.2, Karen Valdez-Gonzalez, M.S.2, Sarah Parisotto M.S.2,
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Felicia Gliksman, M.D.3, Stephen Thompson, M.D.3, Jomard Sabri1, Evan Fertig, M.D.1
Northeast Regional Epilepsy Group, 20 Prospect Avenue, Hackensack, NJ 07601
2
Hackensack University Medical Center, Division of Genetics, Department of Pediatrics, 30
Prospect Avenue, Hackensack NJ 07601 3
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1
Hackensack University Medical Center, Division of Pediatric Neurology, Department of
Title character count: 74
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Pediatrics, 30 Prospect Avenue, Hackensack NJ 07601
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Running title character count: 44 Abstract word count: 253 Main text word count: 1908 Number of tables: 4 Number of figures: 0 References: 12
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Running title: Next-Generation Sequencing in pediatric epilepsy Key words: gene panel, next-generation sequencing, children, epilepsy Correspondence: Eric BJ Ségal, MD Northeast Regional Epilepsy Group 20 Prospect Avenue Suite 801 Hackensack, NJ 07601 Phone: 201-343-6676 Fax: 201-343-6689 Email: [email protected]
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Abstract
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Introduction: When there are no chromosomal variations found, patients with suspected genetic etiologies can be tested using next-generation sequencing(NGS), known as epilepsy panels.
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Objective: The primary objective of this study was to examine the diagnostic yield of nextgeneration sequencing(NGS) seizure panels, known as epilepsy panels, in medication-resistant epilepsy subjects between 0-18 years old in subjects with non clinically-significant comparative genomic hybridization microarray (array-CGH) results. Methods: This is a single-center retrospective chart review of the diagnostic-yield of nextgeneration sequencing(NGS) seizure panels, known as epilepsy panels, in medication-resistant epilepsy subjects between 0-18 years old in subjects with non clinically-significant comparative genomic hybridization microarray(microarray-CGH) results from January 2011 to December 2014. The primary endpoint was the yield of clinically-significant NGS results.
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Results: Forty-nine subjects (21 male) were identified as having medication-refractory epilepsy with non-clinically-significant array-CGH results. NGS abnormalities were seen in 28 subjects(57%): 7 of these subjects(25%) had clinically significant findings. Mutations were found in the SCN1A gene in 3 subjects, in the PCDH19 gene in 2 subjects, and in DLG3 and in MECP2, TSC2, and SLC9A6 genes 1 subject each. Only the MECP2 mutation was found to be pathogenic in this last subject. The additional yield of NGS with uninformative CMA was 14%. Positive findings were primarily seen in those with Dravet Syndrome, all with SCN1A mutations(42% of clinically significant results). Given the small sample size, a larger prospective study would be helpful to determine the clinical yield of NGS.
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Conclusion: NGS seizure panels could be a useful tool in the diagnosis of non-acquired pediatric medication-refractory epilepsy with uninformative array-CGH.
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Introduction
Forty-seven percent of patients with refractory epilepsy have cryptogenic etiologies.i In addition
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to a comprehensive understanding of a patient’s history, examination, electroencephalogram, brain magnetic resonance imaging, many of these patients will undergo genetic testing to further
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elucidate a possible etiology. Genetic testing may include metabolic evaluation, chromosomal analysis, and molecular testing targeting epilepsy.
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In many epilepsy centers, molecular microarray testing such as array comparative genomic hybridization (microarray-CGH) is one of the first-line diagnostic tests in order to understand epilepsy etiology. This diagnostic test can detect abnormalities such as microdeletions,
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microduplications, copy number variants, and regions of homozygosity.ii
When there are no chromosomal variations found, patients with suspected genetic etiologies can be tested using next-generation sequencing(NGS), known as epilepsy panels. NGS provides the ability to analyze multiple genes simultaneously. NGS technologies can more effectively identify monogenomic epilepsies. Different laboratories have established panels of genes that relate primarily to epilepsy.
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The costs of NGS can be prohibitively high and often denied for patients with government-
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subsided insurance. The practice in our Center is to reserve NGS for patients with non-acquired medication-refractory epilepsy. Given the cost and challenge of obtaining the test, it is important to determine the diagnostic yield of NGS in the pediatric epilepsy population. Mercimek-
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Maghmuoglu, et. al. recently studied the diagnostic yield of NGS testing of pediatric epileptic encephalopathy.iii They found a 45% yield of clinically significant NGS abnormalities with 4.5%
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of the subjects had metabolic disorders diagnosed via this molecular testing. Lemke, et al. developed an epilepsy-NGS tool testing 265 genes and tested a pilot population of 33 adults and children of different clinical phenotypes and found a diagnostic yield of 48%.iv The goal of this study is to evaluate the diagnostic yield of NGS in children with non-acquired medication-
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refractory epilepsy who already have non-clinically significant chromosome microarray,
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metabolic, and neuroimaging.
Methods
A retrospective chart review was performed searching for subjects between 0-18 years of age with medication-refractory epilepsy subjects who had genetic testing performed from 2011-2014. Subjects were considered to be medication refractory after poor response to at least two different
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neurologists at Hackensack University Medical Center.
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anti-convulsant medications.v Subjects were referred to the genetics department from the
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Subjects who did not have both chromosomal microarray-CGH and next-generation sequencing panels were excluded.
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Charts were reviewed for results of brain imaging studies, metabolic evaluation, chromosomal microarray-CGH analysis, single gene molecular analysis and NGS panels. Metabolic evaluation included plasma amino acids, urine organic acids and based on clinical phenotype other metabolic tests were performed. This could include urine acyclglycines, carnitine levels,
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and lumbar puncture for neurotransmitters.
The array-CGH were performed by a variety of vendors depending on patient insurance contracts or hospital agreement (Clairsure® 190K probe Quest Diagnostics, Nichols Institute, San Juan
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Capistrano, CA; Affymetrix® 170K probe Laboratory Corporation of America Holdings, Research Triangle Park, NC; Agilent® 180K probe Mayo Medical Laboratories, Rochester, MN;
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Oligonucleotide array Bioreference Laboratories, Elmwood Park, NJ).
Epilepsy NGS testing was performed at either GeneDx (Gaithersburg, MD, USA) or at Courtagen Life Sciences (Woburn, MA, USA). Specific genes sequenced are listed in Table 4. Laboratory selection was mostly based on insurance approval and hospital agreements.
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Demographic information, clinical data related to seizures and epilepsy, and test outcomes were
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extracted by a clinical research assistant and reviewed by board certified genetic counselors, medical geneticist and a pediatric epileptologist. Etiologies for epilepsy were categorized using
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the ILAE 2010 recommendations.vi
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Data included gender, age of onset of seizures, history of developmental delay, first degree family history of seizures or epilepsy, prevalence of dysmorphic features, abnormal neurological exam (other than developmental delay), seizure types and history of abnormal MRIs. Descriptive statistics were utilized to convey the results of the analysis and to quantify the diagnostic yield of
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Results
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each test.
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A total of 49 subjects were identified for this study(Table 1). Twenty-one of the subjects were male. The mean onset of epilepsy was 2.6 (SD±0.6) years old.
Thirty subjects (61%) demonstrated developmental delay. There was a family history of epilepsy in 15 subjects (31%). Ten subjects (20%) had non-neurological medical problems.
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Physical exam findings revealed 2 subjects (4%) had dysmorphic features and 18 subjects (36%)
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had abnormal neurological examinations. Neither of the subjects with dysmorphic features were
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found to have clinically significant NGS results.
Diagnostic testing showed 17 subjects (35%) had abnormal MRI findings. Most of these findings
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consisted of non-specific findings such as partial agenesis of corpus callosum. However, 2 subjects did have malformations of cortical development (i.e. pachygyria and polymicrogyria). Neither of those subjects had any clinically significant molecular testing. None of the subjects
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with clinically significant NGS had abnormal MRIs.
Using the 2010 ILAE recommendations for classification of etiologies, 14 subjects (29%) had presumed genetic, 1 patient (2%) had presumed metabolic, 5 subjects (10%) had structural, and
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29 subjects (59%) had unknown etiologies(Table2).
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Ten subjects (20%) had abnormal, non-clinically significant array-CGH results. NGS abnormalities were seen in 28 subjects (57%). Seven of these subjects (25%) had clinically significant findings (Table 3). Of these 7 subjects, 3 had SCN1A mutations, 2 had PCDH19 mutations, 1 had a DLG3 mutation, and 1 had mutations in the MECP2, TSC2, and SLC9A6 genes. For this final patient, it was determined that only the MECP2 mutation was pathogenic, while the other two variants were found to be non-significant.
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At the time of testing, none of the subjects with clinically-significant NGS results had
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dysmorphism, non-neurological problems, or clinically significant MRI findings.
Subject 1 was 3.5 months old at age of seizure onset. The patient’s seizures were initially focal afebrile seizures. This patient did go on to develop myoclonic seizures at 13 months of age and
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found to have febrile-triggered seizures. Developmental delay was not present. MRI brain
demonstrated minimal nonspecific periventicular white matter T2 intensity as well as subtle
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increased signal intensity of both hippocampi without volume loss.
Subject 2’s seizures began at 14 months of age without febrile seizures. However, the patient later had febrile-induced seizures. The patient had an inconsistent response to sodium-channel agents. For example, oxcarbazepine and lacosamide exacerbated the subject’s seizures and
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lamotrigine did not. The clinical history was significant for developmental delays in the gross motor, fine motor, and speech domains. On exam, the patient was found to have appendicular hypotonia. At a previous institution, prior to NGS testing, this subject underwent a surgical
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evaluation including subdural electrode placement without resection. MRI performed prior to
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surgical evaluation was concerning for a nonspecific linear T2/FLAIR hyperintense signal within the left anterior corona radiate which was deemed an incidental finding. Subject 3’s first seizure was a febrile seizure at 18 months of age. This patient underwent a language regression around the same time as onset of seizures. Like subject 2, the patient has heat-triggered seizures. The patient’s MRI demonstrated a nonspecific foci of T2 prolongation within the left frontal and right parietal subcortical white matter. This finding did not evolve on
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repeat imaging and was deemed an incidental finding. The subject’s father was found to have
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the same genetic mutation.
The positive MECP2 mutation was found in a female who started having multifocal seizures at 5 weeks of life. The patient met criteria for malignant migrating partial epilepsy in infancy. The
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patient underwent genetic testing at that time prior to developing her clinical manifestations including decelerated head growth, developmental delay, and non-purposeful use of her hands.
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Upon presentation, the subject was found to have a normal MRI brain. The subjects exam was significant for axial and appendicular hypotonia.
Subject 6 initially presented with epileptic spasms that evolved to drop seizures. The patient was found to have global developmental delays, axial and appendicular hypotonia, and Autistic
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Spectrum Disorder. The patient’s MRI was normal. Both subjects that tested positive for PCDH19 mutations were female with hypotonia and global
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developmental delay. Subject 4 was also diagnosed with intellectual disability and ADHD.
Discussion
In our population, NGS helped diagnose 14% of children with medication refractory epilepsy. NGS represents the next step in the genetic diagnostic pathway. Previous studies have studied NGS yield in either mixed-age population or children with epileptic encephalopathies. To our
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knowledge, this is the first report of the diagnostic yield of pediatric medication-refractory
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epilepsy who have non-clinically significant array-CGH.
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NGS testing yielded a genetic sequence variant in more than half of all subjects, and of these,
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14% had clinically significant mutations.
The majority of significant results were found in subjects with a SCN1A mutation. This mutation, identified in Dravet Syndrome patients, is beneficial for pharmacologic purposes. There are anti-convulsant medications that are contraindicated and others known to be more
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effective for this specific syndrome.vii Early knowledge of an SCN1A mutation, therefore, can save the patient from unnecessary treatments and hospitalizations. In our cohort, these genetic findings resulted in discontinuation of sodium blocking agents as well as further conversations
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with families regarding prognosis.
Many patients with SCN1A mutations have a phenotype consistent with Dravet Syndrome. This
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would include febrile seizure beginning during the first year of life typically beginning between 5-8 months of age. While some of the subjects did later develop more typical features consistent with Dravet Syndrome, the atypical age of onset of seizures, seizure types, and inconsistent response to sodium-channel agents warranted broader testing.viii Two subjects diagnosed with PCDH19 mutation has led to not only more informative counseling but also possible more targeted therapies. Patients with PCDH19 mutations can have clinical
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histories similar to patients with Dravet Syndrome.ix The genetic diagnosis can open-up further
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therapies.x For example, there are on-going studies using new-medications for this particular epilepsy etiology (clinical trial ID: NCT02358538).
The case of subject 7 highlights the diagnostic advantage of early use of NGS in children with
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medication-refractory epilepsy and previously negative testing. The positive MECP2 mutation was found prior to developing her clinical manifestations including decelerated head growth,
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developmental delay, and non-purposeful use of her hands. With this mutation, the patient also met criteria for the Hanefeld Variant of atypical Rett Syndrome.xi Additionally, to our knowledge, MECP2 mutations have not been described in patients with malignant migrating partial epilepsy of infancy. Although this particular mutation did not change epilepsy
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management, it did help with anticipatory guidance for the family.
Another patient had a positive DLG3 mutation which has been associated with cortical hyperexcitability in the NMDA pathway.xii At the time of diagnosis, there was no specific
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therapy available for mutations involving the NMDA receptor, however, it is conceivable that
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this patient may respond to anti-glutameteric agents such as perampanel. Consideration must be given to the limitations of the study. It is retrospective, based in a single center, and involves a small sample size. In addition, different vendors were used for NGS as well as for array-CGH testing which may lead to further inconsistencies. Larger, prospective studies on children with medication refractory epilepsy would be helpful to determine the clinical yield of NGS.
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This study demonstrates the yield of NGS testing represents the next step in diagnosing children
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with non-acquired medication-refractory epilepsy. This testing leads to more informed
conversations regarding prognosis, family planning, and in some instances more targeted
therapies. As more mechanism-specific agents become available, it is feasible that appropriate
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identification can lead to a more rational and personalized approach of epilepsy treatment.
Conclusion:
Our study demonstrates that NGS can be a clinically useful diagnostic test for establishing an
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etiology for medication-refractory epilepsy and improving accuracy of prognosis and clinical management. Clinicians should consider using NGS in the evaluation of medication refractory epilepsy patients in whom neuroimaging and typical standard genetic testing has not yielded
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clinically significant results.
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Table 1 – Clinical characteristics of all subjects(n=49) 21/28
Mean age of onset of seizures
2.6 (range 0-17 years old)
Developmental delay
30
Family history of seizures/epilepsy
15
Additional medical history
1
Dysmorphic features
2
Abnormal neurological exam
18
Abnormal MRI
17
Abnormal array-CGH
10 28
Clinically significant abnormal NGS
7
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Abnormal NGS
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Boys/Girls
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Total(%) 14 (29%) 1 (2%) 5 (10%) 29 (59%)
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Diagnosis Genetic Metabolic Structural Unknown
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Table 2 – Results of array-CGH and NGS grouped by epilepsy diagnosis Clinicallysignificant NGS(%) 6 (85.7%) 0 1 (14.2%) 0
Abnormal arrayCGH(%) 1 (10%) 1 (10%) 1 (10%) 7 (70%)
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Table 3 – Clinical characteristics Age of Testing (years) 4.5
Developmental MRI Delay Prior to Findings Seizures No Incidental
2
Yes
8
No
PCDH19 PCDH19
Age of Onset (years) Missense 0.25 (febrile) Frameshift 1.2 (afebrile) Missense 1.5 (febrile) Missense 0.6 Missense 0.8
SCN1A
2
SCN1A
3
SCN1A
4 5
13 2.5
Yes Yes
6 7
DLG3 MECP2
Missense Missense
4 0.2
0.6 0.1
Epilepsy Syndrome
Incidental Incidental
Yes Yes
Normal Normal Normal Normal
Febrile GTC, hemiconvulsive afebrile seizures, GTC Febrile seizures, focal dyscognitive Hemi-clonic GTC, myoclonic jerks Spasms Hemi-clonic, dyscognitive
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Table 4 Gene Panels Episeek® (Courtagen)
Seizure Semiology
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1
Type of Mutation
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Gene
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Subject
Genetic Mutations
KANSL1 CNTNAP2, MBD5, NRXN1, SLC9A6, TCF4, UBE3A CHRNA2, CHRNA4, CHRNB2, LGI1
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Koolen-de Vries Syndrome Angelman/Angelman-like/PittHopkins Autosomal Dominant Focal Epilepsies Benign familial infantile seizures Benign familial neonatal seizures Benign familial neonatal-infantile seizures Cerebral folate deficiency Creatine deficiency syndromes EAST/SeSAME syndrome Familial infantile myoclonic epilepsy Generalized epilepsy with febrile seizures Hyperekplexia Joubert Syndrome
Juvenile Myoclonic Epilepsy Migraine Neuronal Ceroid Lipofucsionses Peroxisomal Biogenesis Disorders
PRRT2 KCNQ2, KCNQ3 SCN2A
FOLR1 GAMT, GATM, SLC6A8 KCNJ10 TBC1D24 GARBG2, SCN1A, SCN1B, SCN2A ARHGEF9, GLRA1, GLRB, GPHN, SLC6A5 AHI1, ARL13B, CC2D2A, CEP290, INPP5E, NPHP1, OFD1, RPGRIP1L, TMEM216, TMEM67 BRD2, CACNB4, EFHC1, GABRA1 ATP1A2, BRAF, CACNA1A CLN3,CLN5,CLN6,CLN8,CTSD,CTSF,DNAJC5,KCTD7,MFSD8,PPT1, TPP1 PEX1,PEX2,PEX3,PEX5,PEX6,PEX7,PEX10,PEX12,PEX13,PEX14, PEX16, PEX19,PEX26
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Progressive Myoclonic Epilepsy Ras/MAPK pathway dysregulation Rett/atypical Rett Syndromes Brain or Nervous System Malformations
ASAH1,CSTB,EPM2A,FOLR1,GOSR2,KCTD7,NHLRC1,PRICKLE1, SCARB2 BRAF,HRAS,KRAS,MAP2K1,MAP2K2,NF1,NRAS,PTPN11,RAF1,SOS1, SPRED1
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CDKL5,FOXG1,MBD5,MECP2,MEF2C AHI1,ARFGEF2,ARL13B,ASPM,ATR,BRAF,BUB1B,C12orf57,CASK,CBL,CC2D2A,CC DC88C,CDK5RAP2,CDON,CENPJ,CEP152,CEP290, COL18A1, COL4A1, CPT2, DCLK2, DCX, ELP4, EMX2, EOMES, FGF8, FGFR3, FKRP, FKTN, FLNA, GLI2, GLI3,GPR56,KAT6B,LAMA2,LARGE, LIG4, MCPH1, MED17, MLC1, NHEJ1, NPHP1, OFD1, PAFAH1B1, PAX6, PCNT, PEX7, PIK3CA, PIK3R2, PLA2G6, PNKP, POMGNT1,POMT1, POMT2, QBP1, PTCH1, RAB3GAP1, RARS2, RELN, SERPINI1, SHH, SHOC2, SIX3, SLC25A19, SNAP25, SNAP29, SRPX2, STIL, STRADA, TGIF1, TSEN2,TSEN34, TSEN54,TUBA1A,TUBA8,TUBB2B,VANGL1,VRK1,WDR62, ZIC2 ALG1,ALG11,ALG12,ALG13,ALG2,ALG3,ALG6,ALG8,ALG9,B4GALT1,COG1,COG4, COG5,COG6,COG7,COG8,DDOST, DOLK,DPAGT1, DPM1, DPM3, MAGT1, MGAT2, MOGS, MPDU1, MPI, NGLY1, PGM1, PMM2, RFT1, SLC35A1, SLC35A2, SLC35C1, SRD5A3, TMEM165, TUSC3 AKT3,CNR1,CNR2,CYP2C19,CYP2C9,CYP3A4,CYP3A5,DAGLA,FAAH,GPR55,MGLL ,MTOR
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Congenital Disorder of Glycosylation
Drug Metabolism and Cannabinoid Receptors and Pathways Early onset Epileptic Encephalopathies or Infantile Spasms
ALDH7A1,ATN1,BRAT1,CACNA1A,CACNA1H,CACNB4,CASR,CHD2,CHRNA2,CHR NA4,CHRNB2,CLCN2,CNTN2,CSTB,DEPDC5,EFHC1, EFHC2, PM2A, GABRA, GABRB3, GABRD, GABRG2, GOSR2, GPR98, GRIN1, GRIN2A, GRIN2B, JRK, KCNMA1, KCNQ2, KCNQ3, KCTD7, MBD5, ME2, HLRC1, PCDH19, PRICKLE1, PRICKLE2, PRRT2, SCARB2, SCN10A, SCN11A, SCN1A, SCN1B, SCN2A, SCN3A, SCN3B, SCN4A, SCN4B, SCN5A, SCN7A, SCN8A, SCN9A, SLC2A1,TBC1D24
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Generalized/Myoclonic/Absence Epilepsies/Febrile Seizures
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GABA Receptors
ALDH7A1,ARHGEF9,ARX,ATP6AP2,CDKL5,CNTNAP2,FH,FOXG1,GABRG2,GRIN2 A,GRIN2B,HNRNPU,KCNQ2,KCNT1, LIAS, MAGI2, MEF2C, EDD4L, NRXN1, PCDH19, PLCB1, PNPO, POLG, PRRT2, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SCN9A, SLC25A22, SLC2A1, SLC6A4, SLC9A6, SPTAN1, ST3GAL3, STXBP1, TCF4, TSC1, TSC2, ZEB2 GABBR1,GABBR2,GABRA2,GABRA3,GABRA4,GABRA5,GABRA6,GABRB1, GABRB2,GABRE,GABRG1,GABRG3,GABRP,GABRQ,GABRR1, GABRR2,GABRR3
Mitochondrial Dysfunction
Protein and Carbohydrate Metabolism
Selected inborn errors of metabolism
Storage disease and organelle dysfunction including lysosomal
ADCK3, APTX, BCS1L, C12orf65, COQ2, COQ9, COX10, COX15, DLD, LRPPRC, NDUFA2, NDUFAF6, NDUFS1, NDUFS3, NDUFS4, NDUFS7, NDUFS8, NDUFV1, OPA1, PC, PDHA1, PDHX, PDSS1, PDSS2, POLG, SCO2, SDHA, SURF1, TACO1, VDAC1 ABCC8, ACY1, ADSL, AGA, ALDH4A1, ALDH5A1, ALDH7A1, AMT, ARG1, ATPAF2, BTD, CTSA, DPYD, ETFA, ETFB, ETFDH, FH, FOLR1, FUCA1, GCDH, GCSH, GLDC, GNE, HPD, HYAL1, L2HGDH, MOCS1, MOCS2, NEU1, PANK2, PGK1, PNPO, PRODH, QDPR, SLC17A5, SLC25A15 SLC46A1, SUMF1, SUOX ABAT, ACOX1, ATIC, ATP5A1, ATP7A, BCKDHA, BCKDHB, C12orf65, DBT, DDC, DHCR7, GLUD1, GLUL, HSD17B10, HSD17B4, KCNJ11, MGME1, MMACHC, MTHFR, MTR, MTRR, NDUFA1, PHGDH, PSAT1, SLC19A3, SUCLA2, TMEM70, VDAC1 ARSA, ARSB, ASPA, EIF2B1, EIF2B2, EIF2B3, EIF2B4, EIF2B5, FUCA1, GALC, GALNS, GFAP, GLB1, GNE, GNPTAB, GNPTG, GNS, GUSB, HEXA, HEXB, SNAT,
ACCEPTED MANUSCRIPT Segal-17
IDS, IDUA, MCOLN1, MLC1, NAGLU, NOTCH3, NPC1, NPC2, PLP1, PSAP, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, SDHA, SGSH, SMPD1, SUMF1, TREX1
Syndromic disorders with epilepsy and others
ABCC2, ASPA, ATP1A2, ATP1A3, ATP2A2, BCKDK, CCL2, CLCNKA, CLCNKB, CNTNAP2, CPT1A, DYRK1A, FGD1, FLVCR2, FOXH1, GJD2, GUSB, HCN1, HCN2, HCN3, HCN4, HERC2, IDH2, KCNA1, KCNAB1, KCNJ1, KCNV2, KIAA1279, KMT2D, LBR, LGI1, MAPK10, NDE1, NIPBL, NODAL, RAI1, RBFOX1, NASEH2C, RTTN, SETBP1, SGCE, SLC1A3, SLC4A10, SMC1A, SMC3, SNIP1, ST3GAL5, TBX1, TRPM6, VPS13A, VPS13B, ZEB2
KANSL1 CNTNAP2, MBD5, NRXN1, SLC9A6, TCF4, UBE3A CHRNA2, CHRNA4, LGI1 PRRT2 KCNQ2, KCNQ3 SCN2A FOLR1 GAMT, GATM, KCNJ10 TBC1D24
TE D
Koolen-de Vries Syndrome Angelman/Angelman-like/PittHopkins Autosomal Dominant Focal Epilepsies Benign familial infantile seizures Benign familial neonatal seizures Benign familial neonatal-infantile seizures Cerebral folate deficiency Creatine deficiency syndromes EAST/SeSAME syndrome Familial infantile myoclonic epilepsy Generalized epilepsy with febrile seizures Juvenile Myoclonic Epilepsy Neuronal Ceroid Lipofucsionses Progressive Myoclonic Epilepsy Ras/MAPK pathway dysregulation Rett/atypical Rett Syndromes Brain or Nervous System Malformations Early onset Epileptic Encephalopathies or Infantile Spasms
M AN U
GeneDx –Comprehensive Epilepsy Panel Epilepsy Syndrome Genetic Mutations
SC
RI PT
storage disorders and leukodystrophies
GARBG2, SCN1A, SCN1B, SCN2A
AC C
EP
CACNB4, EFHC1, GABRA1 CLN3, CLN5, CLN6, CLN8, CTSD, DNAJC5, KCTD7, MFSD8, PPT1, TPP1 CSTB,EPM2A,FOLR1,GOSR2,KCTD7,NHLRC1,PRICKLE1, SCARB2 BRAF,HRAS,KRAS,MAP2K1,MAP2K2,NF1,NRAS,PTPN11,RAF1,SOS1, SPRED1
Generalized/Myoclonic/ Absence Epilepsies/Febrile Seizures
Generalized/Myoclonic/Absence
CDKL5,FOXG1,MBD5,MECP2,MEF2C PNKP, SLC25A19, SRPX2 ALDH7A1, ARX,ATP6AP2,CDKL5,CNTNAP2, FOXG1,GABRG2,GRIN2A, GRIN2B, KCNQ2, LIAS, MAGI2, MEF2C, NRXN1, PCDH19, PNPO, POLG, PRRT2, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SLC25A22, SLC2A1, SLC6A4, SLC9A6, SPTAN1, STXBP1, TCF4, TSC1, TSC2, ZEB2 ALDH7A1, CACNB4, CHRNA2, CHRNA4,CHRNB2,CSTB, GABRA, GABRB3, GABRG2, GOSR2, GRIN2A, GRIN2B, KCNQ2, KCNQ3, KCTD7, PRICKLE1, PRRT2, SCARB2, SCN1A, SCN1B, SCN2A, SLC2A1
ALDH7A1, CSTB, GABRD, GABRG2, GOSR2, GRIN1, GRIN2A, GRIN2B, KCNQ2,
ACCEPTED MANUSCRIPT Segal-18
KCNQ3, KCTD7, MBD5, PRICKLE1, PRRT2, SCARB2, SCN1A, SCN1B, SCN8A, SLC2A1
Protein and Carbohydrate Metabolism
ADSL, ALDH7A1, FOLR1, PNPO, SLC25A15
RI PT
Epilepsies/Febrile Seizures
i
ix
TE D
M AN U
SC
Kwan, Patrick, and Martin J. Brodie (2012). Early identification of refractory epilepsy New England Journal of Medicine 342.5 (2000): 314-319. ii Pong, A. W., Pal, D. K., & Chung, W. K. (2011). Developments in molecular genetic diagnostics: an update for the pediatric epilepsy specialist. Pediatric neurology, 44(5), 317-327. iii Lemke, J. R., Riesch, E., Scheurenbrand, et. al, (2012). Targeted next generation sequencing as a diagnostic tool in epileptic disorders. Epilepsia, 53(8), 1387-1398. iv Mercimek-Mahmutoglu, S., Patel, J., Cordeiro, D., et. Al. (2015). Diagnostic yield of genetic testing in epileptic encephalopathy in childhood. Epilepsia, 56(5), 707-716. v Kwan P, Arzimanoglou A, Berg AT, et. Al. (2010). Definition of drug resistant epilepsy: consensus proposal by the ad hoc Task Force of the ILAE Commission on Therapeutic Strategies. Epilepsia 51(6) 1069-77. vi Berg, A. T., Berkovic, S. F., Brodie, M. J., et. Al., (2010). Revised terminology and concepts for organization of seizures and epilepsies: report of the ILAE Commission on Classification and Terminology, 2005–2009. Epilepsia, 51(4), 676-685. vii Chiron, Catherine, and Olivier Dulac. "The pharmacologic treatment of Dravet syndrome." Epilepsia 52.s2 (2011): 72-75. viii Dravet, C (2011). The core Dravet syndrome phenotype. Epilepsia, 52(Suppl.2):3-9.
AC C
EP
Depienne, C., Bouteiller, D., Keren, B., et. Al., (2009). Sporadic infantile epileptic encephalopathy caused by mutations in PCDH19 resembles Dravet syndrome but mainly affects females.PLoS Genet, 5(2), e1000381. x Marini, C., Darra, F., Specchio, N., et. Al. (2012). Focal seizures with affective symptoms are a major feature of PCDH19 gene–related epilepsy. Epilepsia, 53(12), 2111-2119. xi Neul, J. L., Kaufmann, W. E., Glaze, et. Al. (2010). Rett syndrome: revised diagnostic criteria and nomenclature. Annals of neurology, 68(6), 944-949. xii Qu, M., Aronica, E., Boer, K., et. Al. (2009). DLG3/SAP102 protein expression in malformations of cortical development: a study of human epileptic cortex by tissue microarray. Epilepsy research, 84(1), 33-41.