Epilepsy AdvancedSequencing Evaluation

Generalized, Absence, Focal, and Myoclonic Epilepsies

Epileptic Encephalopathy

Neuronal Migration Disorders

X-Linked Intellectual Disability

Neuronal Ceroid Lipofuscinosis

Epilepsy with Migraine

Syndromic Disorders

Infantile Spasms

P H E N O T Y P E - B A S E D N E X T G E N E R A T I O N S E Q U E N C I N G F O R E P I L E P S Y

Testing that Makes a Difference.

A Phenotype-Based Next Generation Sequencing Approach

A T H E N A D I A G N O S T I C S

NEXTGEN TESTING

When your patient presents with symptoms ofepilepsy, the specific cause is not always clear. Using Next Generation Sequencing (NGS) touncover a genetic cause of disease may lead toanswers that direct the most appropriate treatment and care for your patient.

Athena Diagnostics now offers NGS evaluations forepilepsy arranged by clinical phenotype, testing onlythe clinically relevant genes, which streamlines thediagnostic process and saves time and money. By combining proprietary NGS technology, fast turnaround time, and Athena Insight™, Athena Diagnostics offers a uniquely powerfuldiagnostic toolset for epilepsy that can make animportant difference in the care of your patient andpredict the risk of disease in family members.

Phenotype-based evaluations forepilepsy harness the power of NGS allowing for earlier diagnosisand teatment , sooner than ever before.

The More You Know, The More You Can Do

Accurate and early diagnosis of epilepsy is essential for both evaluation and treatment because specific interventions are increasingly being recognized.1

The Value of Genetic Testing • Identify the variant responsible for disease,

clarify the diagnosis, and help guide patienttreatment and care decisions

• Save the patient and family from a prolongedseries of stressful, invasive, and expensivediagnostic procedures

• Reveal the possible genetic cause for thepatient’s symptoms or those of their familymembers

• Help counsel the patient and relativesconcerning possible recurrence risk

• Provide key genetic information for futureplanning - a positive or a negative test result canhave implications for both the patient and thefamily

Matthew Gallen, Associate Scientist

1. Phenotype-Guided Evaluations• Athena Diagnostics Epilepsy Advanced Sequencing Evaluations

are organized by phenotype, making test selection easy and avoiding costly and unnecessary testing.

• Evaluations are based on peer-reviewed published literature to facilitate clinically-meaningful diagnostic and treatment decisions.

2. Fast, Cost-Efficient Testing with Powerful Proprietary Technology• Concordance with Sanger sequencing, the “gold standard”

for sequence quality, is achieved for excellent sensitivity and specificity.

• Our hybridization-based enrichment technology allows removalof duplicate fragments, reducing amplification bias, providingexcellent allele balance, more confident variant calls, and better sensitivity; our use of long baits provides excellent specificity.

• Regions that would otherwise exhibit low coverage are boosted with an additional proprietary bait library process developed by Athena Diagnostics scientists and geneticists.

• Regions with known pseudogenes or other sequence homology are sequenced at no additional charge using Sanger technology if interference with NGS accuracy is detected.

• Each evaluation is accomplished with one blood draw and 6-week turnaround.

3. Athena Insight™, In-Depth Variant Interpretation and Reporting• Created by scientists and geneticists at Athena Diagnostics, Athena Insight™ incorporates a standardized,

evidence-based pathogenicity assessment/scoring process that stratifies variants into seven categoriesbased on the relative likelihood of pathogenicity.

• Our team of variant scientists collaborate on evaluations to provide the best possible results and optimal analytic value.

• Assessments of variants are based on multiple independent types of evidence. This provides the confidenceof pathogenicity assessment from accumulated evidence in the medical literature, as well as variant databases.

• The clinician receives a complete synopsis and interpretation of findings with a determination of thelikelihood of variants being benign or pathogenic. Results are presented in clear, concise terms suitable for use during discussions with patients and family members.

• Over 14,000 pathogenicity assessments on more than 10,000 unique variants have been successfullyperformed to date, focused on genetic disorders in neurology, endocrinology, and nephrology. This allows amore accurate definition of the region of interest.

• In many cases, it is beneficial to test other family members to enhance interpretations of variants of unknownsignificance. Please call an Athena Diagnostics genetic counselor for details.

Phenotype-Based NGSDiagnostics

Fast, Cost-efficient

NGS Technology

In-DepthResults

Interpretation

ATHENADIAGNOSTICS

NEXTGENTESTING

Three Reasons Why Next Generation Sequencing fromAthena Diagnostics Can Make a Difference

ClinicalEvaluation

Assignmentof

Phenotype

Diagnostics

Treatment

Follow-up/Monitoring

Includes neurologicalhistory, physical exam,electroencephalogram(EEG), neuroimaging.2

Typically identifiable on the basis of the age of onset, seizuretype, specificelectroencephalogram(EEG) and neuroimagingcharacteristics, andthorough clinicalevaluation.

A phenotype-basedNGS diagnosticapproach allowsclinicians tocharacterize a patient's epilepsy into defined categories anduncover clinically-relevant geneticcauses of disease.

Specificantiepileptictherapies can berecommendedbased on thecausativevariant.

Antiepileptic drugmonitoring isavailable fromQuest Diagnostics.

Continuum of Care for Epilepsy IncludingPhenotype-Based Next Generation Sequencing

Comprehensive Servicesthat Go Beyond Results

Genetic Counseling Services

Our team of genetic counselors is readily available to providein-depth information on the nature, inheritance, and implications of genetic test results.

• Genetic counseling can help the physician guide thepatient in making informed medical and personaldecisions for themselves and their family.

Pre-Authorization Services

Pre-authorization for in-network orders eases the workload inyour office.

Care360® The power and flexibility of this cloud-based, electronichealth record system developed by Quest Diagnosticsenables 24/7 access and greater efficiencies in managing lab orders and results.

• Place test orders, analyze results, and gain practice-wide insights quickly and easily.

• Save time and improve access to critical patient information.

Carol A. Hoffman, Ph.D., M.S., LGC Genetic Counselor

Phenotype-Based Next Generation Sequencing for Epilepsy

Epilepsy Genes Tested

5001 5002 5003 5004 5005 5006 5007 5008 5000Generalized Epileptic Neuronal X-Linked Neuronal Epilepsy Syndromic Infantile Epilepsy AdvancedAbsence, Encephalopathy Migration Intellectual Ceroid with Disorders Spasms Sequencing EvaluationFocal and Disorders Disability Lipofuscinosis MigraineMyoclonicEpilepsies

ALDH7A1 ARHGEF9 ARFGEF2 ARHGEF9 CLN3 ATP1A2 ATP2A2 ARX ALDH7A1 FKRP OPHN1 SLC9A6CACNA1A ARX ARX ARX CLN5 CACNA1A ATP6V0A2 CDKL5 ARFGEF2 FKTN PAFAH1B1 SMC1A

CASR CDKL5 COL18A1 ATP6AP2 CLN6 NOTCH3 CCDC88C FOXG1 ARHGEF9 FLNA PAK3 SMC3CHRNA2 CNTNAP2 COL4A1 ATRX CLN8 POLG CLCNKA GABRB3 ARX FOXG1 PANK2 SMSCHRNA4 FOXG1 CPT2 CASK CTSD PRRT2 CLCNKB GRIN2A ATP1A2 GABRA1 PAX6 SNAP29CHRNB2 GABRG2 DCX CDKL5 DNAJC5 SCN1A* KCNA1 MEF2C ATP2A2 GABRB3 PCDH19 SPTAN1

CSTB GRIN2A EMX2 CUL4B KCTD7 SLC2A1 KCNJ1 SCN2A ATP6AP2 GABRD PEX7 SRPX2DEPDC5 KCNT1 FGFR3 DCX MFSD8 KCNJ10 SLC25A22 ATP6V0A2 GABRG2 PHF6 STXBP1EFHC1 MECP2 FKRP FGD1 PPT1 KIAA1279 SPTAN1 ATRX GPC3 PIGV SYNGAP1EPM2A NRXN1 FKTN GPC3 TPP1 LBR STXBP1 CACNA1A GPR56 PLA2G6 SYPGABRA1 PCDH19 FLNA GRIA3 LGI1 CASK GRIA3 PLP1 TBC1D24GABRB3 PNKP GPR56 HSD17B10 MLL2 CASR GRIN2A PNKP TBX1GABRD RNASEH2A LAMA2 KDM5C NIPBL CCDC88C HSD17B10 POLG TCF4GABRG2 RNASEH2B LARGE MECP2 PANK2 CDKL5 KCNA1 POMGNT1 TPP1GRIN2A RNASEH2C PAFAH1B1 OFD1 PIGV CHRNA2 KCNJ1 POMT1 TREX1KCNMA1 SAMHD1 PAX6 OPHN1 PLA2G6 CHRNA4 KCNJ10 POMT2 TSC1KCNQ2 SCN1A* PEX7 PAK3 RAI1 CHRNB2 KCNMA1 PPT1 TSC2KCNQ3 SCN1B POMGNT1 PCDH19 SERPINI1 CLCNKA KCNQ2 PQBP1 TUBA1AKCNT1 SCN2A POMT1 PHF6 SETBP1 CLCNKB KCNQ3 PRICKLE1 TUBA8KCTD7 SCN8A POMT2 PLP1 SMC3 CLN3 KCNT1 PRICKLE2 TUBB2BLGI1 SCN9A PQBP1 PQBP1 SYNGAP1 CLN5 KCTD7 PRRT2 UBE3A*

MBD5 SLC25A22 RAB3GAP1 RAB39B TBX1 CLN6 KDM5C RAB39B VPS13AME2 SLC2A1 RELN SLC9A6 TSC1 CLN8 KIAA1279 RAB3GAP1 VPS13B

NHLRC1 SLC9A6 SNAP29 SMC1A TSC2 CNTNAP2 LAMA2 RAI1 WDR62PCDH19 SPTAN1 SRPX2 SMS VPS13A COL18A1 LARGE RELN ZEB2PRICKLE1 STXBP1 TUBA1A SRPX2 VPS13B COL4A1 LBR RNASEH2APRICKLE2 SYNGAP1 TUBA8 SYP CPT2 LGI1 RNASEH2BPRRT2 TCF4 TUBB2B CSTB MBD5 RNASEH2CSCARB2 TREX1 WDR62 CTSD ME2 SAMHD1SCN1A* UBE3A* CUL4B MECP2 SCARB2SCN1B ZEB2 MEF2C SCN1A*SCN2A DCX MFSD8 SCN1BSCN9A DEPDC5 MLL2 SCN2ASLC2A1 DNAJC5 NHLRC1 SCN8A

SLC4A10 EFHC1 NIPBL SCN9ATBC1D24 EMX2 NOTCH3 SERPINI1

EPM2A NRXN1 SETBP1FGD1 OFD1 SLC25A22FGFR3 SLC2A1

SLC4A10

A T H E N A D I A G N O S T I C S

NEXTGEN TESTING

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

GENERALIZEDMYOCLONIC/ABSENCEEPILEPSIES, FEBRILE

SEIZURES, FOCAL SEIZURES

• Myoclonic seizures • Absence seizures("Petit Mal")

• Tonic seizures • Clonic seizures • Tonic-clonic seizures("Grand Mal")

• Atonic seizures• Febrile seizures• Focal seizures• Electroclinical syndromes characterized by recognizable entities such as childhood absence epilepsy

• Complexes of clinical features, signs, and symptoms that together define a distinctive, recognizable clinical disorder of generalizedseizures6

5001, Epilepsy Advanced Sequencing Evaluation-Generalized, Absence, Focal, and Myoclonic Epilepsies

36 Genes Tested

• Genetic Epilepsy with FebrileSeizures Plus (GEFS+)

• Dravet syndrome • Unverricht-Lundborg disease

• Lafora disease • Benign neonatal seizures • Progressive myoclonicepilepsy

• Nocturnal frontal lobeepilepsy

• Susceptibility to childhoodabsence epilepsy

• Benign familial infantileseizures

• Action myoclonus-renal failure syndrome (AMRF)

• Epilepsy with neuro-developmental defects

• Pyridoxine-dependentepilepsy

• Familial focal epilepsy with variable foci

EPILEPTICENCEPHALOPATHIES

• High probability of encephalopathic features that presentor worsen afterepilepsy onset

• Developmental delayor regression relativeto same-age peers

• May occur at any agebut most commonand severe in infancyand early childhoodwhere global and profound cognitiveimpairment mayoccur

• Often pharmaco-resistant6

5002, Epilepsy Advanced SequencingEvaluation-EpilepticEncephalopathy

31 Genes Tested

• Early infantile epileptic encephalopathies

• Rett syndrome • GEFS+ • Pitt-Hopkins-like syndrome

• Aicardi-Goutieres syndrome 1-5

• Angelman syndrome • Epilepsy and Mowat-Wilson syndrome

• Cortical dysplasia-focal epilepsy syndrome

• Christianson syndrome

• Severe epileptic encephalopathy with autonomic dysfunction

NEURONAL MIGRATIONDISORDERS

• Severe cognitivedeficits and epilepsyrecognized primarilyin childhood

• Brain malformations observed on neuroimaging

• Microcephaly7

5003, Epilepsy Advanced SequencingEvaluation-NeuronalMigration Disorders

29 Genes Tested

• Lissencephaly • Periventricular heterotopia

• Periventricular heterotopia with microcephaly

• Schizencephaly • Congenital musculardystrophy, dystro-glycanopathy withbrain and eye anomalies types A1-3

• Cerebral malformations and Warburg micro syndrome

• Polymicrogyria, symmetric or asymmetric

• Rolandic epilepsy • Polymicrogyria withoptic nerve hypoplasia

EPILEPSY IN X-LINKED

INTELLECTUAL DISABILITY

• Clinical and EEGspectrum of seizuretypes range fromfever-inducedseizures only, to recalcitrant hypsarrhythmia-associated seizures

• Seizures accompany but do not cause developmental delay

• May be de novo or have X-linked inheritance pattern8,9

5004, Epilepsy Advanced SequencingEvaluation-X-Linked Intellectual Disability

27 Genes Tested

• X-linked epilepsywith intellectual disability (several subtypes)

• Seizure disorder associated with X-linked intellectualdisability (severalsubtypes)

• Christianson syndrome

• Epilepsy and intellectual disabilityrestricted to females

• Rett syndrome • Rolandic seizures • West syndrome, X-linked infantilespasms

• Seizures in Pelizaeus-Merzbacher disease

• Early infantile epilep-tic encephalopathy 1,2,8,9

PHEN

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Athena Diagnostics Phenotype-based Testing for the Causes of EpilepsyTest selection is intuitive - conveniently arranged by clinically relevant groups of epilepsy phenotypes. This approach is especially useful in diagnosing cases with broad phenotypes and non-specific clinical findings.

NEURONALCEROID

LIPOFUSCINOSIS

• Mental and motor deterioration

• Epilepsy • Visual loss• Ataxia • Accumulation of autofluorescent storage materialupon tissue biopsy

• Variable age of onset-from congenital toadult

• Mostly autosomal recessive inheritance10

5005, Epilepsy Advanced SequencingEvaluation-NeuronalCeroid Lipofuscinosis

10 Genes Tested

• Congenital NeuronalCeroid Lipofuscinosis(NCL)

• Infantile NCL• Late infantile NCL• Variant late infantileNCL

• Juvenile NCL• Adult NCL (Kufs disease)

EPILEPSYWITH

MIGRAINE

• Epilepsy and/or migraine that canprecede or succeedeach other or occursimultaneously

• Predisposition forepilepsy: EEG can measure pre-disposition forseizures and showlack of habituationfor migraine

• Repeated episodes ofparoxysmal events,e.g.: seizures orheadache, often preceded by prodromes and/orauras11

5006, Epilepsy Advanced SequencingEvaluation-Epilepsywith Migraine

7 Genes Tested

• Familial hemiplegic migraine 1-3 including familialhemiplegic migrainewith progressivecerebellar ataxia

• Familial basilar migraine

• Migraine associatedwith mitochondrialrecessive ataxia syndrome (includessensory ataxic neuropathy,dysarthria, and ophthalmoparesis(SANDO) and spinocerebellarataxia with epilepsy(SCAE)

SYNDROMIC DISORDERS WITH

EPILEPSY

• Variable and complexphenotypes that in-clude epilepsy12

5007, Epilepsy Advanced SequencingEvaluation-Syndromic Disorders

26 Genes Tested

• Intractable seizures,focal cortical dysplasia,Taylor balloon cell type

• Tuberous sclerosis • SESAME syndrome • Episodic ataxia/myokymia syndrome

• Darier disease• Congenital hydrocephalus

• Bartter syndrome• Cohen syndrome• Goldberg-Shprintzenmegacolon syndrome

• Pelger-Huet anomaly• Kabuki syndrome• Cornelia de Lange syndrome

• Infantile neuroaxonal dystrophy

• Schinzel-Giedion midface retraction syndrome

• DiGeorge syndrome• Choreoacanthocytosis

INFANTILE SPASMS

• Clusters of epilepticflexor/extensorspasms with ictalelectrodecrement

• Age of onset usually before 1 year

• West syndrome (infantile spasms,hypsarrhythmia, developmental delay)

• May be accompaniedby cognitive impair-ment, autism, andmovement disorders5

5008, Epilepsy Advanced SequencingEvaluation- Infantile Spasms

10 Genes Tested

• West syndrome • Infantile spasm syndrome, X-linked

• Infantile spasms associated with Rett syndrome, congenitalvariant

• Early-onset epilepticspasms associatedwith epilepsy withneuro-developmentaldefects

• Infantile spasms associated with intellectual disability,stereotypic move-ments, and/or cerebral malformations

• West syndrome associated with earlyinfantile epileptic encephalopathy3,4,5

The organization of Athena Diagnostics Evaluations is based on evidence from publications using NGS as a diagnostic tool forepileptic disorders and relevant variant databases.3,4,5

* Data on file.

EPILEPSY OFGENETIC ORIGIN

• May include non-specific phenotypes

• Characteristics can include those in the eight subpanelslisted.

5000, Epilepsy Advanced SequencingEvaluation (Includes5001, 5002, 5003,5004, 5005, 5006,5007, 5008)

141 Genes Tested

• Disorders/phenotypesin the eight subpanelslisted.

Whole Blood, Test Test CPT Lavender Top Tube/ TurnaroundCode Name Code(s)* Minimum Volume** Time

5001 Epilepsy Advanced Sequencing Evaluation - 81403(1), 81404(3), 81405(6), 10 mL 6 weeksGeneralized, Absence, Focal, and Myoclonic 81406(3), 81407(1), 81479(1)Epilepsies, 36 Genes Tested

5002 Epilepsy Advanced Sequencing Evaluation - 81302(1), 81404(3), 81405(4), 10 mL 6 weeks Epileptic Encephalopathy, 31 Genes Tested 81406(6), 81407(1), 81479(1)

5003 Epilepsy Advanced Sequencing Evaluation - 81404(3), 81405(3), 81406(4), 10 mL 6 weeks Neuronal Migration Disorders, 29 Genes Tested 81407(1), 81408(2), 81479(1)

5004 Epilepsy Advanced Sequencing Evaluation - 81302(1), 81404(1), 81405(4), 10 mL 6 weeks X-Linked Intellectual Disability, 81406(2), 81407(1), 81479(1) 27 Genes Tested

5005 Epilepsy Advanced Sequencing Evaluation - 81479(1) 10 mL 6 weeks Neuronal Ceroid Lipofuscinosis, 10 Genes Tested

5006 Epilepsy Advanced Sequencing Evaluation - 81405(1), 81406(3), 81407(1), 10 mL 6 weeks Epilepsy with Migraine, 7 Genes Tested 81479(1)

5007 Epilepsy Advanced Sequencing Evaluation - 81404(2), 81405(1), 81406(2), 10 mL 6 weeks Syndromic Disorders, 26 Genes Tested 81407(1), 81408(1), 81479(1)

5008 Epilepsy Advanced Sequencing Evaluation - 81404(2), 81406(2), 81479(1) 10 mL 6 weeks Infantile Spasms, 10 Genes Tested

5000 Epilepsy Advanced Sequencing Evaluation - 81302(1), 81403(1), 81404(9), 10 mL 6 weeks (Includes all Subpanels: 5001, 5002, 5003, 81405(12), 81406(18), 81407(4), 5004, 5005, 5006, 5007, 5008), 141 Genes Tested81408(3), 81479(1)

65 ARX Duplication/Deletion Test (Epilepsy) 81403(1) 10 mL 28 days

67 CDKL5 Duplication/Deletion Test (Epilepsy) 81405(1) 10 mL 28 days

537 SCN1A Deletion Test 81479(1) 10 mL 21-28 days

410 CSTB (EPM1) DNA Test 81404(1) 10 mL 28 days

797 ARX Evaluation (Epilepsy) 81403(1), 81404(1) 10 mL 28 days

799 CDKL5 Evaluation (Epilepsy) 81045(1), 81406(1) 10 mL 28 days

674 CSTB (EPM1) Evaluation 81404(2) 10 mL 28 days

573 SCN1A Evaluation 81407(1), 81479(1) 10 mL 21-28 days

521 TSC1 DNA Sequencing Test 81406(1) 10 mL 28 days

508 TSC1 Deletion Test 81405(1) 10 mL 21-28 days

522 TSC2 DNA Sequencing Test 81407(1) 20 mL 28 days

524 TSC2 Deletion Test 81406(1) 10 mL 28 days

556 Complete Tuberous Sclerosis Evaluation 81405(1), 81406(2), 81407(1) 10 mL 4-6 weeks

*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 DNAthat meet 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 oneof our genetic counselors regarding specific acceptance policies and specimen requirements for prenatal testing at 800-394-4493.

References: 1. Muthugovindan D, Hartman AL. Pediatric Epilepsy Syndromes. Neurologist 2010;16:223-37. 2. Fisher RS, Maslah S. How is Epilepsy Diagnosed? EpilepsyTherapy Project. Stanford. Available from: www.epilepsy.com. Accessed January 6, 2014. 3. Lemke JR, Riesch E, Scheurenbrand T, et al. Targeted next generation sequencingas a diagnostic tool in epileptic disorders. Epilepsia 2012;53:1387-98. 4. Garofalo S, Cornacchione M, Di Costanzo A. From Genetics to Genomics of Epilepsy. Neurol ResearchInternational 2012;2012:ID876234. 5. Paciorkowski AR, Thio LL, Dobyns WB. A genetic and biologic classification of infantile spasms. Pediatric Neurol. 2011;45:355-67. 6. 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 andTerminology, 2005-2009. Epilepsia 2010;51:676-85. 7. Liu JS. Molecular Genetics of Neuronal Migration Disorders. Curr Neuro Neurosci Rep. 2011;11:171-178. 8. Stevenson RE,Holden KR, Rogers RC, Schwartz CE. Seizures and X-linked intellectual disability. Eur J Med Gen. 2012; 55:307-12. 9. Vincent AK, Noor A, Janson A, et al.  Identification ofgenomic deletions spanning the PCDH19 gene in two unrelated girls with intellectual disability and seizures. Clin Genet 2012 Dec; 82(6) :540-5. 10. Kousi M, Lehesjoki AE,Mole SE. Update of the Mutation Spectrum and Clinical Correlations of over 360 Mutations in Eight Genes that Underlie the Neuronal Ceroid Lipofuscinoses. Hum Mutat.2012;33:42-63. 11. Bianchin MM, Londero RG, Lima JE, Bigal ME. Migraine and Epilepsy: A Focus on Overlapping Clinical, Pathophysiological, Molecular, and TherapeuticAspects. Curr Pain Headache Rep. 2010;14:276-83. 12. Merwick A, O'Brien M, Delanty N. Complex single gene disorders and epilepsy. Epilepsia 2012;53 Suppl 4:81-91.

©2014 Athena Diagnostics, the Athena Diagnostics logo and Recombx are registered trademarks of Athena Diagnostics, Inc.Athena Insight is a trademark of Athena Diagnostics, Inc. Unless otherwise noted, any person depicted in this material is a model. ADX537SG-1/14JS-REV00

Testing that Makes a Difference.

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