Phenotype-based Neurology Next-Generation Sequencing with Copy Number Variant Analysis

Generalized, Absence, Focal, Febrile, and Myoclonic Epileptic Encephalopathy Developmental Brain Malformations Intellectual Disability Neuronal Ceroid Lipofuscinosis Epilepsy with Migraine Syndromic Disorders Infantile Spasms

Now with 234 Getting a clearer picture of the causes of epilepsy

Harnessing the power of NGS for earlier diagnosis Athena Diagnostics offers a comprehensive approach to diagnosing the causes of epilepsy, which may be helpful when considering different treatment options and avoiding contraindicated drugs. Our phenotype-based approach is supported by clinical diagnostic exome sequencing to provide molecular diagnosis for a significant proportion of patients with epilepsy.11 The benefits include:

• An accurate diagnosis sooner at typically less cost Phenotype- When your patient presents with epilepsy based NGS than traditional methods testing with CNV analysis symptoms, the specific cause is not always • Reduced turnaround time (TAT) clear. Using next-generation sequencing • More informed future Athena Diagnostics (NGS) to uncover a genetic cause of planning for the patient NextGen and their family Fast, testing In-depth disease may lead to answers that can cost-efficient results NGS technology interpretation aid in identifying the most appropriate treatment and care for your patient.

® Athena Diagnostics provides NGS A comprehensive panel delivering actionable insights evaluations for epilepsy arranged by clinical Athena Diagnostics Epilepsy Advanced Sequencing and CNV phenotype, testing the most comprehensive Evaluations feature: collection of clinically-relevant genes • Next-generation sequencing and CNV testing for 234 genes known to be associated with epilepsy, • Phenotype-guided evaluations, making test selection easier and which streamlines the diagnostic process may help avoid costly, unnecessary testing and may save time and money. • NGS technology for excellent sensitivity and specificity, supported by a proprietary bait library process to boost results • A single blood draw, with a 28-35 day turnaround By combining NGS technology, copy number • Athena Insight™, providing in-depth variant interpretation and variant (CNV) analysis, fast turnaround reporting, fully backed by a dedicated team of experts to ensure time, and Athena Insight™, Athena optimal analytic value Diagnostics offers a uniquely powerful • Genetic counselors and medical directors available to provide clinicians with in-depth information on the nature, inheritance, diagnostic toolset for epilepsy that can and implications of genetic test results, as well as guidance on make an important difference in the care test selection of your patient and can help predict the risk of disease in family members.

Accurate and early diagnosis of epilepsy syndrome, when possible, is essential for both evaluation and treatment because specific antiepileptic therapies are increasingly recognized.1 Phenotype-based testing for the causes of epilepsy

At Athena Diagnostics, we’re committed to helping you and nonspecific clinical findings. The organization of deliver efficient, quality healthcare. That’s why our test these panels is based on evidence from publications using selection is intuitive — conveniently arranged by clinically- NGS as a diagnostic tool for epileptic disorders and relevant relevant groups of disease phenotypes. This approach is variant databases.2,3,4 especially useful in diagnosing cases with broad phenotypes

Tested phenotypes and characterizations

Epilepsy Advanced Sequencing and CNV Evaluations Epilepsy Advanced Sequencing and CNV Evaluations

6008 6010 6018 6019 6022 6023 6033 6038 6000 Generalized, Absence, Epileptic Developmental Intellectual Neuronal Ceroid Epilepsy Syndromic Infantile Epilepsy Advanced Focal, Febrile and Encephalopathy Brain Malformations Disability Lipofuscinosis with Migraine Disorders Spasms Sequencing and CNV Myoclonic Epilepsies Evaluation 84 genes 67 genes 40 genes 56 genes 13 genes 7 genes 31 genes 16 genes 234 genes

• Myoclonic seizures • High probability of encephalopathic • Severe cognitive deficits and epilepsy • Clinical and EEG spectrum of seizure • Mental and motor • Epilepsy and/or migraine that • Variable and complex • Clusters of epileptic flexor/ • May include nonspecific • Absence seizures (“Petit Mal”) features that present or worsen after recognized primarily in childhood types range from fever-induced deterioration can precede or succeed each phenotypes that include extensor spasms with ictal phenotypes epilepsy onset seizures only, to recalcitrant other or occur simultaneously epilepsy11 electrodecrement • Tonic seizures • Brain malformations observed on • Epilepsy • Characteristics can • Developmental delay or regression neuroimaging hypsarrhythmia-associated seizures • Predisposition for epilepsy: • Age of onset usually before include those in the 8 • Clonic seizures • Visual loss relative to same-age peers 6 • Seizures accompany but do not cause EEG can measure pre- 1 year subpanels listed • • Ataxia • Tonic-clonic seizures (“Grand Mal”) • Often pharmaco-resistant5 developmental delay disposition for seizures • West syndrome (infantile • Accumulation of and show lack of habituation • Atonic seizures • May occur at any age but most common • May be de novo or have X-linked spasms, hypsarrhythmia, 7,8 autofluorescent storage for migraine • Febrile seizures and severe in infancy and early inheritance pattern developmental delay) material upon tissue biopsy • Repeated episodes of • Focal seizures childhood, when global and profound • May be accompanied by cognitive impairment may occur • Variable age of onset, from paroxysmal events, e.g.: cognitive impairment, , • Electroclinical syndromes congenital to adult seizures or headache, often and movement disorders4 characterized by recognizable entities • Mostly autosomal recessive preceded by prodromes and/or 10 such as childhood absence epilepsy inheritance9 auras • Complexes of clinical features, signs, and symptoms that together define a distinctive, recognizable clinical disorder of generalized seizures5 The right genes

The Athena Diagnostics team of neurologists and genetic relevant genes to identify a genetic cause. Subpanels are counselors have researched and selected genes associated constructed based on primary phenotype. All 234 genes with primary epilepsy, as well as secondary syndromes known include NGS sequencing, copy number variant analysis, to include seizures. This provides the clinician with the most and chromosomal microarray confirmation if a CNV is found.

Tested phenotypes and characterizations

Epilepsy Advanced Sequencing and CNV Evaluations Epilepsy Advanced Sequencing and CNV Evaluations

6008 6010 6018 6019 6022 6023 6033 6038 6000 Generalized, Absence, Epileptic Developmental Intellectual Neuronal Ceroid Epilepsy Syndromic Infantile Epilepsy Advanced Focal, Febrile and Encephalopathy Brain Malformations Disability Lipofuscinosis with Migraine Disorders Spasms Sequencing and CNV Myoclonic Epilepsies Evaluation 84 genes 67 genes 40 genes 56 genes 13 genes 7 genes 31 genes 16 genes 234 genes

• Myoclonic seizures • High probability of encephalopathic • Severe cognitive deficits and epilepsy • Clinical and EEG spectrum of seizure • Mental and motor • Epilepsy and/or migraine that • Variable and complex • Clusters of epileptic flexor/ • May include nonspecific • Absence seizures (“Petit Mal”) features that present or worsen after recognized primarily in childhood types range from fever-induced deterioration can precede or succeed each phenotypes that include extensor spasms with ictal phenotypes epilepsy onset seizures only, to recalcitrant other or occur simultaneously epilepsy11 electrodecrement • Tonic seizures • Brain malformations observed on • Epilepsy • Characteristics can • Developmental delay or regression neuroimaging hypsarrhythmia-associated seizures • Predisposition for epilepsy: • Age of onset usually before include those in the 8 • Clonic seizures • Visual loss relative to same-age peers 6 • Seizures accompany but do not cause EEG can measure pre- 1 year subpanels listed • Microcephaly • Ataxia • Tonic-clonic seizures (“Grand Mal”) • Often pharmaco-resistant5 developmental delay disposition for seizures • West syndrome (infantile • Accumulation of and show lack of habituation • Atonic seizures • May occur at any age but most common • May be de novo or have X-linked spasms, hypsarrhythmia, 7,8 autofluorescent storage for migraine • Febrile seizures and severe in infancy and early inheritance pattern developmental delay) material upon tissue biopsy • Repeated episodes of • Focal seizures childhood, when global and profound • May be accompanied by cognitive impairment may occur • Variable age of onset, from paroxysmal events, e.g.: cognitive impairment, autism, • Electroclinical syndromes congenital to adult seizures or headache, often and movement disorders4 characterized by recognizable entities • Mostly autosomal recessive preceded by prodromes and/or 10 such as childhood absence epilepsy inheritance9 auras • Complexes of clinical features, signs, and symptoms that together define a distinctive, recognizable clinical disorder of generalized seizures5

Comprehensive services and solutions that go beyond results

A team of medical experts Convenient preauthorization services We have developed a staff of board-certified geneticists Preauthorization for in-network orders eases the workload and neurologists, genetic counselors, doctors, scientists, in your office. and researchers who see themselves as an extension of your team. Choose Athena Diagnostics and take advantage of: Personalized patient services • On-call or appointment-based consultation and support • Support from point of order to specimen collection • Economical testing approaches driven by algorithmic to submission and appeals efficiency • Regionally structured to provide local expertise • The latest discoveries and insights into the newest genetic testing technologies

Next-generation sequence testing for epilepsy NGS and CNV analysis performed on all genes

6008 6010 6018 6019 6022 6023 6033 6038 6000 Generalized, Epileptic Develop­ Intellectual Neuronal Epilepsy Syndromic Infantile Epilepsy Advanced Absence, Focal, Enceph­ mental Disability Ceroid Lipo­ with Disorders Spasms Sequencing and CNV Evaluation Febrile and Myoc­ alopathy Brain Mal­ 56 Genes fuscinosis Migraine 31 Genes 16 Genes 234 Genes lonic Epilepsies 67 Genes formations 13 Genes 7 Genes 84 Genes 40 Genes

ABAT HCN4 ADSL ARFGEF2 ABAT ATP13A2 ATP1A2 ADGRV1 ADSL ABAT DYRK1A LMNB2 SCN3A ADSL KCNC1 ALG13 ARX ADSL CLN3 CACNA1A ANKRD11 ARX ADGRV1 EEF1A2 MAGI2 SCN5A ALDH7A1 KCNH2 ALG9 ASPM ALG13 CLN5 NOTCH3 ATP2A2 CACNA2D1 ADSL EFHC1 MBD5 SCN8A ALG13 KCNMA1 AMT CENPJ ALG9 CLN6 POLG ATP6V0A2 CDKL5 ALDH7A1 EMX2 MCPH1 SCN9A ALG9 KCNQ2 ARHGEF9 COL4A1 ARHGEF9 CLN8 PRRT2 GFAP FOXG1 ALG13 EPM2A MECP2 SERPINI1 ALPL KCNQ3 ARX CPT2 ARX CTSD SCN1A* HPRT1 GABRB3 ALG9 FGD1 MEF2C SETBP1 AMT KCNT1 BRAT1 DCX ATP6AP2 CTSF SLC2A1 KANSL1 GRIN2A ALPL FGFR3 MFSD8 SETD2 ASAH1 KCTD7 CACNA2D1 DEAF1 ATRX DNAJC5 KCNA1 MEF2C AMT FKRP NDE1 SHH ASPM L2HGDH CACNA2D2 DPYD BCKDK GRN KCNJ10 NR2F1 ANKRD11 FKTN NDUFA1 SIK1 ATP1A3 LGI1 CDKL5 EMX2 CACNA2D1 KCTD7 KIAA1279 SCN2A ARFGEF2 FLNA NHLRC1 SIX3 BCKDK LIAS CHD2 FGFR3 CASK MFSD8 KMT2D SLC25A22 ARHGEF9 FOLR1 NIPBL SLC13A5 BRAT1 LMNB2 CNTNAP2 FKRP CDKL5 PPT1 LBR SLC35A2 ARX FOXG1 NOTCH3 SLC19A3 CACNA1A MBD5 DNM1 FKTN CHRNA7 TPP1 LGI1 SPTAN1 ASAH1 GABRA1 NR2F1 SLC25A19 CACNA1H NDUFA1 FLNA CUL4B MAGI2 ST3GAL3 ASPM GABRB2 NRXN1 SLC25A22 CACNB4 NHLRC1 EEF1A2 FOLR1 DCX NIPBL STXBP1 ATP13A2 GABRB3 OFD1 SLC2A1 CASR PCDH19 FOXG1 GPR56 DEAF1 PANK2 TBL1XR1 ATP1A2 GABRD OPHN1 SLC35A2 CHD2 PHGDH GABRG2 LAMA2 DPYD PIGA ATP1A3 GABRG2 PAFAH1B1 SLC4A10 CHRNA2 PIGO GLDC LARGE1 DYRK1A PIGN ATP2A2 GAMT PAK3 SLC6A1 CHRNA4 PNPO GNAO1 MCPH1 EEF1A2 PIGV ATP6AP2 GATM PANK2 SLC6A8 CHRNA7 PRICKLE1 GRIN1 NDE1 FGD1 PLA2G6 ATP6V0A2 GFAP PAX6 SLC9A6 CHRNB2 PRICKLE2 GRIN2A PAFAH1B1 FOLR1 RAI1 ATRX GLDC PCDH19 SMC1A CPA6 PRIMA1 GRIN2B PAX6 GABRB2 ROGDI BCKDK GNAO1 PEX7 SMC3 CRH PRRT2 HCN1 PEX7 GAMT SERPINI1 BRAT1 GOSR2 PHF6 SMS CSTB SCARB2 HNRNPU POMGNT1 GATM SETBP1 CACNA1A GPC3 PHGDH SNAP25 CYP27A1 SCN1A* KCNA2 POMT1 GFAP SMC3 CACNA1H GPR56 PIGA SPATA5 DEPDC5 SCN1B KCNB1 POMT2 GPC3 SYNGAP1 CACNA2D1 GRIA3 PIGN SPTAN1 DYNC1H1 SCN2A KCNJ11 PQBP1 GRIA3 TBX1 CACNA2D2 GRIN1 PIGO SRPX2 EFHC1 SCN3A KCNT1 QARS GRIN2B TSC1 CACNB4 GRIN2A PIGV ST3GAL3 EPM2A SCN5A LIAS RAB3GAP1 HNRNPU TSC2 CASK GRIN2B PLA2G6 ST3GAL5 FOLR1 SCN9A MECP2 RELN HSD17B10 VPS13A CASR GRN PLCB1 STIL GABRA1 SLC19A3 NRXN1 SHH IQSEC2 VPS13B CDKL5 HCN1 PLP1 STX1B GABRB2 SLC25A19 PCDH19 SIX3 KDM5C CENPJ HCN4 PNKP STXBP1 GABRB3 SLC2A1 PIGA SLC25A19 KIAA2022 CHD2 HNRNPU PNPO SUCLA2 GABRD SLC35A2 PIGN SRPX2 MECP2 CHRNA2 HPRT1 POLG SYN1 GABRG2 SLC4A10 PLCB1 STIL OFD1 CHRNA4 HSD17B10 POMGNT1 SYNGAP1 GAMT SLC6A1 PNKP TSEN54 OPHN1 CHRNA7 IQSEC2 POMT1 SYNJ1 GATM SLC6A8 PNPO TUBA1A PAK3 CHRNB2 KANSL1 POMT2 SYP GLDC ST3GAL5 PURA TUBA8 PCDH19 CLN3 KCNA1 PPT1 SZT2 GOSR2 STX1B QARS TUBB2B PHF6 CLN5 KCNA2 PQBP1 TBC1D24 GRIN2A SUCLA2 RNASEH2A WDR62 PLP1 CLN6 KCNB1 PRICKLE1 TBL1XR1 GRIN2B SYNJ1 RNASEH2B PQBP1 CLN8 KCNC1 PRICKLE2 TBX1 HCN1 TBC1D24 RNASEH2C PURA CNTNAP2 KCNH2 PRIMA1 TCF4 SAMHD1 RAB39B COL4A1 KCNJ10 PRRT2 TPP1 SCN1A* RBFOX1 CPA6 KCNJ11 PURA TREX1 SCN1B SETD2 CPT2 KCNMA1 QARS TSC1 SCN2A SLC35A2 CRH KCNQ2 RAB39B TSC2 SCN8A SLC6A8 CSTB KCNQ3 RAB3GAP1 TSEN54 SCN9A SLC9A6 CTSD KCNT1 RAI1 TUBA1A SIK1 SMC1A CTSF KCTD7 RBFOX1 TUBA8 SLC13A5 SMS CUL4B KDM5C RELN TUBB2B SLC19A3 SNAP25 CYP27A1 KIAA1279 RNASEH2A UBE3A SLC25A19 SPATA5 DCX KIAA2022 RNASEH2B VPS13A SLC25A22 SRPX2 DEAF1 KMT2D RNASEH2C VPS13B SLC2A1 SYN1 DEPDC5 L2HGDH ROGDI WDR45 SLC35A2 SYP DNAJC5 LAMA2 SAMHD1 WDR62 SLC9A6 WDR45 DNM1 LARGE1 SCARB2 WWOX SPTAN1 DOCK7 LBR SCN1A* ZEB2 ST3GAL5 DPYD LGI1 SCN1B STXBP1 DYNC1H1 LIAS SCN2A SYNGAP1 SZT2 TCF4 TREX1 UBE3A WDR45 WWOX ZEB2

*Includes parental testing. Parental results will only be reported in the context of the proband.

Next-generation sequence testing for epilepsy Comprehensive test menu for epilepsy from Athena Diagnostics

Whole blood, Test Turnaround Test name CPT code(s)* lavender-top tube/ code time min. volume (mL)** 81403(3), 81404(12), 81405(17), Epilepsy Advanced Sequencing and CNV Evaluation 6000 81406(20), 81407(8), 81408(3), 8 mL 28–35 days (234 genes) 81479(1) Epilepsy Advanced Sequencing and CNV Evaluation— 81403(1), 81404(4), 81405(6), 6008 Generalized, Absence, Focal, and Myoclonic Epilepsies 8 mL 28–35 days 81406(4), 81407(4), 81479(1) (84 genes) 81302(1), 81304(1), 81403(2), Epilepsy Advanced Sequencing and CNV Evaluation— 6010 81404(4), 81405(6), 81406(6), 8 mL 28–35 days Epileptic Encephalopathy (67 genes) 81407(1), 81479(1) 81403(1), 81404(4), 81405(4), Epilepsy Advanced Sequencing and CNV Evaluation— 6018 81406(4), 81407(2), 81408(2), 8 mL 28–35 days Developmental Brain Malformations (40 genes) 81479(1) 81302(1), 81304(1), 81403(1), Epilepsy Advanced Sequencing and CNV Evaluation— 6019 81404(3), 81405(6), 81406(2), 8 mL 28–35 days Intellectual Disability (56 genes) 81407(1), 81479(1) Epilepsy Advanced Sequencing and CNV Evaluation— 6022 81406(1), 81479(1) 8 mL 28–35 days Neuronal Ceroid Lipofuscinosis (13 genes) Epilepsy Advanced Sequencing and CNV Evaluation— 81405, 81406(3), 81407(2), 6023 8 mL 28–35 days Epilepsy with Migraine (7 genes) 81479 Epilepsy Advanced Sequencing and CNV Evaluation— 81404(1), 81405(3), 81406(2), 6033 8 mL 28–35 days Syndromic Disorders (31 genes) 81407(2), 81408(1) 81479 Epilepsy Advanced Sequencing and CNV Evaluation— 81403, 81404(2), 81405, 6038 8 mL 28–35 days Infantile Spasms (16 genes) 81406(2), 81479 1129 SCN1A Sequencing and CNV Evaluation 81407(1), 81479(1) 8 mL 28–35 days 1191 SCN1A CNV Test 81479(1) 8 mL 28–35 days Complete Sequencing and CNV 1131 81405(1), 81406(2), 81407(1) 8 mL 28–35 days Evaluation 1245 TSC1 Sequencing Test 81406(1) 8 mL 28–35 days 1255 TSC2 Sequencing Test 81407(1) 8 mL 28–35 days 1236 TSC1 CNV Test 81405(1) 8 mL 28–35 days 1254 TSC2 CNV Test 81406(1) 8 mL 28–35 days 1036 ARX Sequencing and CNV Evaluation (Epilepsy) 81403(1), 81404(1) 8 mL 28–35 days 1115 CDKL5 Sequencing and CNV Evaluation (Epilepsy) 81401, 81404 8 mL 28–35 days 1133 CSTB (EPM1) Sequencing and Repeat Expansion Evaluation 81404(2) 8 mL 28–35 days 1003 GFAP (Alexander Disease) Sequencing Test 81405(1) 8 mL 28–35 days

*The CPT codes provided are based on AMA guidelines and are for informational purposes only. CPT coding is the sole responsibility of the billing party. **We require whole blood in EDTA tubes (lavender-top tubes). Adults: 10 mL; children: 4 mL; infants: 2 mL. Outside DNA is discouraged; however, high-quality extracted DNA that meets our standards can be accepted. The test requires a minimum of 20 ug of DNA at a concentration of 50 ng/ul with a minimum volume of 400 ul. Please contact one of our genetic counselors regarding specific acceptance policies and specimen requirements for prenatal testing at 800.394.4493. For more information about genetic or autoimmune testing for epilepsy, contact your Athena Diagnostics representative or visit us online at AthenaDiagnostics.com/Epilepsy. Client services representatives are available from 8:30 AM to 6:30 PM eastern time (U.S.). Customers in the U.S. and Canada please call toll free 1.800.394.4493.

References 1. Muthugovindan D, Hartman AL. Pediatric Epilepsy Syndromes. Neurologist 2010;16:223-37. 2. Garofalo S, Cornacchione M, Di Costanzo A. From Genetics to Genomics of Epilepsy. Neurol Research International 2012;2012:ID876234. 3. Paciorkowski AR, Thio LL, Dobyns WB. A genetic and biologic classification of infantile spasms.Pediatric Neurol. 2011;45:355-67. 4. Berg AT, Berkovic SF, Brodie MJ, et al. Revised terminology and concepts for organization of seizures and epilepsies: Report of the ILAE Commission on Classification and Terminology, 2005-2009. Epilepsia 2010;51:676-85. 5. Liu JS. Molecular Genetics of Neuronal Migration Disorders. Curr Neuro Neurosci Rep. 2011;11:171-178. 6. Stevenson RE, Holden KR, Rogers RC, Schwartz CE. Seizures and X-linked intellectual disability. Eur J Med Gen. 2012; 55:307-12. 7. Vincent AK, Noor A, Janson A, et al. Identification of genomic deletions spanning the PCDH19 in two unrelated girls with intellectual disability and seizures. Clin Genet 2012 Dec; 82(6) :540-5. 8. Kousi M, Lehesjoki AE, Mole SE. Update of the Spectrum and Clinical Correlations of over 360 in Eight Genes that Underlie the Neuronal Ceroid Lipofuscinoses. Hum Mutat. 2012;33:42-63. 9. Bianchin MM, Londero RG, Lima JE, Bigal ME. Migraine and Epilepsy: A Focus on Overlapping Clinical, Pathophysiological, Molecular, and Therapeutic Aspects. Curr Pain Headache Rep. 2010;14:276-83. 10. Merwick A, O’Brien M, Delanty N. Complex single gene disorders and epilepsy. Epilepsia 2012;53 Suppl 4:81-91. 11. Helbig, et al. Diagnostic exome sequencing provides a molecular diagnosis for a significant proportion of patients with epilepsy. 2016. GIM. 18 (9). 12. Fisher RS, Maslah S. How is Epilepsy Diagnosed? Epilepsy Therapy Project. Stanford. Available from: www.epilepsy.com. Accessed January 6, 2014. AthenaDiagnostics.com ©2017 Athena Diagnostics, Inc. Athena Diagnostics and the Athena Diagnostics logo are registered trademarks of Athena Diagnostics, Inc. Athena Insight is a trademark of Athena Diagnostics, Inc. Quest, Quest Diagnostics, any associated logos, and all associated Quest Diagnostics registered or unregistered trademarks are the property of Quest Diagnostics. Unless otherwise noted, any person depicted in this material is a model. ADX537SG-3/17-REV03