The Epidemiology and Aetiologies of the Severe Epilepsies of Infancy

THE EPIDEMIOLOGY AND AETIOLOGIES OF THE SEVERE EPILEPSIES OF INFANCY

Katherine Brooke Howell

MBBS (Hons) B Med Sci FRACP

orcid.org/0000-0002-5469-8411

Submitted in total fulfilment of the requirements of the degree of

Doctor of Philosophy

December 2016

Department of Paediatrics

Faculty of Medicine, Dentistry and Health Sciences

The University of Melbourne

Abstract

The severe epilepsies of infancy (SEI) are a group of infantile-onset seizure disorders characterised by frequent seizures, abnormal EEG and pharmacoresistance to antiepileptic therapy. SEI include well-described epilepsy syndromes such as early infantile epileptic encephalopathy (EIEE), epilepsy of infancy with migrating focal seizures (EIMFS) and West syndrome. Cognitive outcome is often poor, due to effects of seizures, the underlying aetiology, and antiepileptic drugs (AEDs) on the developing brain.

There is an urgent need for novel treatments. Where effective therapies are available, such as epilepsy surgery for brain malformations, treatment can be life-changing. Given that developmental outcomes may be significantly improved in the context of optimal seizure control at an early age, determining the underlying cause of SEI early in life is paramount.

The aetiologies of SEI are heterogeneous; a large number of acquired and genetic brain disorders are reported. In many infants, the cause remains unknown despite investigation, and is presumed to be genetic. With the emergence of next generation sequencing (NGS) techniques such as whole exome sequencing (WES), a rapidly growing number of genetic causes of SEI is now recognised and gene discovery is ongoing. The genetic epidemiology of SEI has not been studied and the relative importance of each genetic cause is not known.

Brain malformations, chromosomal abnormalities, inborn errors of metabolism and some genetic disorders can be diagnosed with technologies currently available in clinical practice. Studies of WES and other NGS techniques in epilepsy populations have shown that these techniques identify the aetiology in 10-50% of undiagnosed patients. No study has specifically looked at the yield in SEI, and no population-based studies have been reported. The yield and cost-effectiveness of NGS for SEI at a population level remains unknown, and access to genetic testing is currently poor in most regions of the world.

This population-based study of SEI in Victoria, Australia aimed to study the incidence and determine the aetiologies, electroclinical phenotypes and other phenotypic characteristics of SEI. As part of a particular focus on genetic aetiologies, the study aimed to identify genetic causes in infants with SEI of unknown aetiology using WES,

and determine the yield and cost of early genetic testing relative to current standard diagnostic pathways for investigation of SEI aetiology.

Infants with SEI born in Victoria during 2011-2013 were identified by a comprehensive state-wide search of multiple sources. Infants with potential SEI were identified by review of all electroencephalogram (EEG) reports on children under two years old during 2011-2015 (n=4505), and search of neonatal intensive care unit (NICU) databases for neonates with seizures born 2011-2013 (n=379). Hospital records of infants with potential SEI from the three main paediatric hospitals in Victoria, The Royal Children’s Hospital (RCH), Monash Health (MMC) and The Austin Hospital, and the two NICUs not co-located with a paediatric hospital, The Royal Women’s Hospital (RWH) and The Mercy Hospital for Women (MHW), were reviewed to confirm clinical and demographic inclusion criteria were met. SEI was defined as epilepsy onset before age 18 months, frequent seizures (> daily for a week or > weekly for a month), epileptiform EEG and pharmacoresistance (failed 2 appropriate antiepileptic therapies); infantile spasms were automatically included. In infants with confirmed SEI, medical records, EEG recordings and brain magnetic resonance imaging (MRI) were reviewed to determine each infant’s epileptic syndrome, outcome at two year old, and aetiology. Clinical assessment and WES were performed if aetiology or electroclinical phenotype was unknown.

114 infants with SEI were ascertained. The incidence of SEI in Victoria is 51/100,000 live births/year. West syndrome/infantile spasms was the most common epileptic syndrome, with an incidence of 33/100,000 live births/year. EIMFS and EIEE had incidences of 4.5 and 3.6/100,000 live births/year respectively.

At two years old, 18 (16%) infants were deceased. 86/98 (90%) survivors had delayed development, and 46/98 (47%) ongoing seizures. All infants whose presenting epileptic syndrome was EIEE, early myoclonic encephalopathy (EME) or EIMFS at epilepsy onset were deceased or had severe developmental impairment. Normal development was seen in 9/64 (14%) infants who presented with West syndromes/infantile spasms or a unifocal epilepsy, and only two were deceased.

The aetiology was identified in 76 (67%) and unknown in 38 (33%). Fourteen (12%) infants had an acquired brain insult such as hypoxic-ischaemic encephalopathy or perinatal stroke. The remaining infants had genetic or presumed genetic aetiologies. Brain malformations were identified in 31 (27%), including focal cortical dysplasia iii

(FCD) in 14 (12%). Six (5%) infants had metabolic disorders and nine (8%) had chromosomal abnormalities. Sixteen (14%) had single gene disorders, including 11 (10%) with disorders of ion channel function (channelopathies).

Aetiology was known from clinical testing in 61 (54%). Research MRI review identified the cause in a further 4 (4%) and research genetic testing in 11 (10%). Among 86 (75%) infants with no aetiological diagnosis prior to epilepsy onset, the highest yield investigations were MRI and genetic testing, which identified the cause in 25/85 (29%) and 16/50 (32%) respectively. 13/50 (26%) infants had a genetic variant of unknown significance (VOUS) identified on WES; these are being further investigated.

Modelling of diagnostic pathways showed that performing WES early in the diagnostic pathway and reducing the amount of metabolic testing increases diagnostic yield for less cost compared with the current standard diagnostic pathway.

Approximately 1:2000 infants have SEI, equating to over 150 new cases of SEI in Australia per year. Outcomes for seizure control, development and survival are poor. Brain malformations were the most common cause, were under-recognised, and should be considered in those with unknown aetiology, especially in those with West syndrome or unifocal epilepsy. Channelopathies were the most common group of single gene disorders. Next-generation genetic testing and high quality brain imaging improved diagnostic yield, with implications for treatment and reproductive counselling, and should be implemented early in the diagnostic pathway in clinical practice.

Future work will focus on identifying the aetiology in the remaining infants, determining the genetic basis of brain malformations causing SEI, and studying the yield of other NGS and brain imaging techniques to improve the rate of early diagnosis. This work will inform research into development of novel and targeted treatments for these devastating disorders.

Declaration of originality

This thesis comprises only my original work unless duly acknowledged as collaborative research. To the best of my knowledge and belief, the thesis contains no material published or written by any person, except where due acknowledgement is given in the text. The thesis contains no material which has been accepted for any other degree in any university. The thesis is less than 100,000 words in length, exclusive of tables, figures, bibliographies and appendices.

Signed ………………………………………………..

Katherine Brooke Howell

Preface

Funding

I am very grateful for funding provided for this thesis. Investigator funding was provided by the Gustav Nossal Postgraduate Scholarship from the National Health and Medical Research Council and the Clifford PhD Scholarship from the University of Melbourne. Funding for WES was provided by an internal grant from the Murdoch Childrens Research Institute (MCRI).

Collaborations

Genetic testing in this study was performed by or with collaborators. WES was performed in collaboration with the Translational Genomics Unit at the MCRI, headed by Dr Stefanie Eggers. Further detail of that collaboration is provided in Chapter 3. Molecular inversion probe-based multigene panel testing was performed by the Mefford lab at the University of Washington, Seattle. Single gene testing of the TBC1D24 gene was performed by Dr Mark Corbett at the University of Adelaide. A small number of patients had research genetic testing via other studies; pathogenic variants identified in these studies are noted in Chapter 5.

I was the joint first author of a review of the genetics of severe epilepsies of infancy and childhood, published in Lancet Neurology in 2015 (McTague and Howell et al). One figure (Figure 1.2) and one table (Appendix A) have been adapted from the paper for this thesis. These adaptations are acknowledged at the point that they appear in the thesis.

Acknowledgements

I am grateful to many people for their contributions to this thesis.

First and foremost, my supervisors. Dr Simon Harvey has been a wonderful primary supervisor. His intellectual input and guidance really enhanced the scientific quality of this study. His efforts extended far beyond that though. I can’t thank him enough for his mentorship, wisdom, support and selflessness, both with regards to this research and personally, during the course of this PhD.

Professor Ingrid Scheffer has brought fantastic enthusiasm to this study, and has taught me so much about genetic epilepsies. She has provided me with invaluable opportunities to get to know people in my field, and be involved in international collaborations and conferences. I am very grateful.

My advisory committee, A/Professor David Amor and Professor Kathryn North, provided honest, valuable and independent guidance on the direction and methods of this study.

I would like to particularly note a major contribution from Professor North, who enabled the grant that funded WES, allowing that aspect of the study to proceed. Dr Leanne Mills and Professor Andrew Sinclair were also integral in facilitating the funding and personnel for WES. I am very grateful for this significant support.

Many clinical colleagues, including neurologists, neurology fellows, EEG scientists, neonatologists, neonatal nurses and a neuroradiologist were involved in this study; without their contributions, this study could not have proceeded. Each person listed here provided support and information, efficiently and cheerfully, for sometimes time- consuming tasks, which is greatly appreciated.

Victorian neurologists and neurology fellows, Dr Simon Harvey, A/Prof Andrew Kornberg, Prof Monique Ryan, Dr Jeremy Freeman, A/Prof Mark Mackay, A/Prof Richard Leventer, Dr Eppie Yiu, Dr Michael Hayman, Dr Victoria Rodriguez-Casero, Dr Gabriel Dabscheck, Dr Eunice Chan, Dr Erik Andersen, A/Prof Michael Fahey, Dr Lindsay Smith, Dr John Archer, Dr Julie Panetta and Ingrid Scheffer provided EEG report data and patient clinical information, and facilitated recruitment of infants to the assessment phase of the study. A/Prof Michael Fahey, A/Prof Mark Mackay, Dr Eppie Yiu, Dr Michael Hayman, Dr John Archer, Dr Julie Panetta, A/Prof Paul Talman and

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Professor Ingrid Scheffer provided support at sites other than RCH, making the process of accessing records very smooth. Catherine Bailey, Sue Watson, Ann Saunders, Giosi Cardamone, Jacqui Christianz, Maria Brusco and James Dahler provided EEG reports. A/Prof Rod Hunt, A/Prof Sue Jacobs, A/Prof Flora Wong, Dr Jim Holberton, Liz Perkins, Kaye Bawden, Rose Li and Tanya Fletcher provided data from neonatal unit databases and medical records. A/Prof Simone Mandelstam reviewed a large number of MRI scans with me, and taught me a lot about interpreting brain imaging in infants.

There are a number of collaborators who performed or supported genetic testing for this study. Dr Stefanie Eggers, Jessica Riseley, A/Prof Sue White, A/Prof Heather Mefford, Dr Gemma Carvill, Dr Candace Myers, Dr Mark Corbett, Ingrid Scheffer, Jacinta McMahon and Amy Schneider all made important contributions, and were very gracious in sharing their knowledge as well as data.

The health economics aspects of this study could not have been performed without health economist Dr Kim Dalziel, clinical costing analyst Malathi Jeremiah and Microsoft excel whiz Justin Green. A/Prof Susan Donath provided helpful advice about studying epidemiology and reporting incidence data.

Prof Sam Berkovic was integral to the choice of topic for this thesis, providing wisdom and insight into areas of need and the practicalities of a larger study, and was a source of guidance throughout the study.

A/Prof Andrew Kornberg, Prof Monique Ryan, A/Prof Rick Leventer, A/Prof Mark Mackay and Dr Eppie Yiu were very generous with their time, mentorship, advice and support during the course of this PhD.

To my husband Justin and our daughter Anna, I am so thankful for all your love, support and patience while I completed this PhD. I can’t wait to be able to join you for weekend bike adventures again.

Finally, I would like to acknowledge and thank all the patients and families who gave so generously of their time to participate in this study. I hope that this work, and future research that follows from it, will begin to make a difference to the lives of infants with severe epilepsies.

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Publications from this and related work

S Balestrini, M Milh, C Castiglioni, K Lüthy, MJ Finelli, P Verstreken, A Cardon, MD, B Gnidovec Stražišar, J Lloyd Holder Jr, G Lesca, MM Mancardi, AL Poulat, GM Repetto, S Banka, L Bilo, LE Birkeland, F Bosch, K Brockmann, JH Cross, D Doummar, TM Félix, F Giuliano, M Hori, I Hüning, H Kayserili, U Kini, MM Lees, G Meenakshi, L Mewasingh, AT Pagnamenta, S Peluso, A Mey, GM Rice, JA Rosenfeld, JC Taylor, MM Troester, CM Stanley, D Ville, M Walkiewicz, A Falace, A Fassio, JR Lemke, S Biskup, J Tardif, NF Ajeawung, A Tolun, M Corbett, J Gecz, Z Afawi, KB Howell, KL Oliver, SF Berkovic, IE Scheffer, FA de Falco, PL Oliver, P Striano, F Zara, PM Campeau, SM Sisodiya. TBC1D24 genotype-phenotype correlation: epilepsies and other neurological features. Neurology, 2016. EPub Jun 8

Howell KB, Harvey AS, Archer JS. Epileptic encephalopathy: Use and misuse of a clinically and conceptually important concept. Epilepsia, 2016; 57:343-347

#McTague A, #Howell KB, Cross JH, Kurian MA, Scheffer IE. The genetic landscape of the epileptic encephalopathies of infancy and childhood. Lancet Neurology, 2015; 15:304-316 #These authors contributed equally

Howell KB, McMahon JM, Carvill GL, Tambunan D, Mackay MT, Rodriguez-Casero V, Webster R, Clark D, Freeman JK, Calvert S, Olson HE, Mandelstam S, Poduri A, Mefford HC, A Simon Harvey, Scheffer IE. SCN2A encephalopathy: a major cause of epilepsy of infancy with migrating focal seizures. Neurology, 2015; 85:958-966

Gemma L. Carvill, Douglas E. Crompton, Brigid M. Regan, Jacinta M. McMahon, Julia Saykally, Matthew Zemel, Amy L. Schneider, Leanne Dibbens, Katherine B. Howell, Simone Mandelstam, Richard J. Leventer, A. Simon Harvey, Saul A. Mullen, Samuel F. Berkovic, Joseph Sullivan, Ingrid E. Scheffer, and Heather C. Mefford. Epileptic spasms are a feature of DEPDC5 mTORopathy. Neurology Genetics, 2015; 1:e17

Marques I, Sa M, Soares G, Mota C, Carvalho C, Aguiar L, Amado M, Delgardo C, Calado A, Dias P, Sousa A, Fortuna A, Santos R, Howell K, Ryan M, Leventer R, Sachdev R, Catford R, Friend K, Mattiske T, Shoubridge C, Jorge P. Unravelling the

pathogenesis of ARX polyalanine tract variants using a clinical and molecular interfacing approach. Molecular Genetics and Genomic Medicine, 2015; 3:203-214

Ware TL, Earl J, Salomons GJ, Struys EA, Peters H, Howell KB, Pitt J, Freeman JL. Typical and atypical phenotypes of PNPO deficiency with elevated CSF and plasma pyridoxamine on treatment. Dev Med Child Neurol 2014; 56:498-502

Epi4K Consortium et al (authors incl. Howell KB). De novo mutations in epileptic encephalopathies. Nature 2013: 501:217-221

Carvill GL, Heavin SB, Yendle SE, McMahon JM, Cook J, Khan A, O’Roak BJ, O’Dorschner M, Calvert S, Malone S, Wallace G, Stanley T, Bye AME, Bleasel A, Howell KB, Kivity S, Mackay MT, Rodriguez-Casero V, Webster R, Smith L, Korczyn A, Afawi Z, Zelnick M, Masalha R, Lerman-Sagie T, Moller RS, Gill D, Andrade D, Freeman JL, Sadleir LG, Shendure J, Berkovic SF, Scheffer IE and Mefford HC. Targeted resequencing in epileptic encephalopathies reveals marked genetic heterogeneity and novel genes including CHD2 and SYNGAP1. Nature Genetics, 2013.

Honors received in the course of this work

Scholarships

National Health and Medical Research Council Gustav Nossal Postgraduate Scholarship 2013-2015 awarded to the highest-ranked clinical applicant for NHMRC PhD scholarships in Australia

University of Melbourne Clifford PhD Scholarship 2014-2015 awarded to the highest-ranked clinician undertaking postgraduate research training at Melbourne Children’s Campus

Prizes

Epilepsy Society of Australia Annual Scientific Meeting 2016 Best Platform award

Table of Contents Abstract ...... ii Declaration of originality...... vi Preface ...... vii Acknowledgements ...... viii Publications from this and related work ...... ix Honors received in the course of this work ...... xi Table of Contents ...... xii List of Tables ...... xvii List of Figures ...... xx Abbreviations ...... xxi

Chapter 1: Literature review ...... 1

1.1 Introduction ...... 2 1.2 Description of the infantile epilepsies ...... 2 1.3 Neurobiology of infantile epilepsies ...... 5 1.4 Clinical features of infant epilepsies ...... 8 1.4.1 Epileptic features ...... 8 1.4.2 Non-epileptic features ...... 25 1.4.3 Survival ...... 31 1.5 Aetiologies of the infantile epilepsies ...... 32 1.5.1 Classification of aetiologies ...... 32 1.5.2 Acquired aetiologies ...... 33 1.5.3 Genetic and presumed genetic aetiologies ...... 34 1.5.4 Advances in knowledge following identification of genetic causes of infantile epilepsies ...... 43 1.5.5 Aetiologies by epileptic syndrome ...... 48 1.5.6 Aetiology by non-epileptic features ...... 59 1.5.7 Infantile epilepsies of unknown aetiology ...... 59 1.5.8 Implications of identifying aetiology ...... 60 1.6 Investigating aetiology of ‘severe’ epilepsies of infancy ...... 60 1.6.1 Current approach ...... 60 1.6.2 Genetic investigation ...... 64 1.6.3 Future of diagnostic investigation ...... 67 1.6.4 Cost of diagnostic investigation ...... 67 1.7 Epidemiology of infantile epilepsies ...... 68 1.7.1 Considerations in interpretation of epidemiologic studies of epilepsy .... 68 xii

1.7.2 Incidence of infantile epilepsies ...... 70 1.8 Burden of infantile epilepsies ...... 74 1.9 What are the severe epilepies of infancy? ...... 75 1.9.1 Terms used to denote ‘severe’ epilepsies ...... 75 1.9.2 Factors that could be considered in a novel definition of ‘severe’ epilepsy of infancy ...... 79 1.10 Conclusions ...... 84

Chapter 2: Introduction, aims and hypotheses ...... 87

2.1 Introduction ...... 87 2.2 Aims ...... 88 2.3 Hypotheses ...... 88

Chapter 3: Methods ...... 89

3.1 Introduction ...... 89 3.2 Study population ...... 91 3.3 Inclusion criteria ...... 93 3.4 Ascertainment ...... 96 3.4.1 Ascertainment Sources ...... 96 3.4.2 Search strategy ...... 98 3.4.3 Confirmation of SEI diagnosis ...... 99 3.5 Assessment and analysis of individual patient data ...... 99 3.5.1 Medical record review ...... 100 3.5.2 Review of seizures and EEG studies ...... 102 3.5.3 Brain MRI analysis ...... 107 3.5.4 Other diagnostic investigations ...... 108 3.5.5 Research clinical assessment...... 108 3.5.6 Genetic testing ...... 109 3.6 Data documentation ...... 114 3.7 Analysis of group data ...... 114 3.7.1 Analysis of search strategies ...... 114 3.7.2 Epidemiology including genetic epidemiology ...... 114 3.7.3 Clinical characteristics and aetiologies ...... 114 3.7.4 Health economics ...... 115 3.7.5 Study time frames ...... 126 3.8 Ethics approvals ...... 127

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Chapter 4: Ascertainment and epidemiology ...... 131

4.1 Ascertainment ...... 131 4.1.1 Ascertainment sources, search strategies and confirmation of SEI diagnosis ...... 131 4.1.2 Discussion ...... 138 4.2 Epidemiology ...... 146 4.2.1 Incidence of SEI ...... 146 4.2.2 Discussion ...... 147 4.3 Conclusions ...... 149

Chapter 5: Aetiologies ...... 151

5.1 Aetiologies ...... 151 5.1.1 Individual aetiologies ...... 151 5.1.2 Aetiologic groups ...... 153 5.1.3 Genetic basis of malformative and metabolic conditions ...... 156 5.1.4 Genetic aetiologies in infants with similarly affected siblings ...... 158 5.1.5 Unknown aetiologies ...... 158 5.1.6 Commentary ...... 159 5.2 Incidence of aetiologies ...... 166 5.2.1 Commentary ...... 166

Chapter 6: Clinical features ...... 169

6.1 Demographics ...... 169 6.2 Epilepsy ...... 169 6.2.1 Commentary on methodology ...... 169 6.2.2 Age of onset ...... 172 6.2.3 Time to presentation ...... 173 6.2.4 Seizure types ...... 173 6.2.5 EEG ...... 175 6.2.6 Seizure frequency ...... 176 6.2.7 Epileptic syndromes ...... 176 6.2.8 Seizure treatment and outcome ...... 183 6.2.9 Commentary ...... 192 6.3 Development ...... 200 6.3.1 Pre-seizure onset ...... 200 6.3.2 Developmental plateau and regression ...... 200 6.3.3 Developmental outcome ...... 200

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6.3.4 Commentary ...... 206 6.4 Comorbidities ...... 209 6.4.1 Commentary ...... 211 6.5 Survival ...... 211 6.5.1 Commentary ...... 213

Chapter 7: Diagnostic investigation ...... 215

7.1 Timing, method and yield of diagnosis ...... 215 7.1.1 Diagnosis prior to seizure onset ...... 216 7.1.2 Diagnosis after seizure onset: standard clinical testing ...... 217 7.1.3 Diagnosis after seizure onset: research imaging review ...... 217 7.1.4 Diagnosis after seizure onset: research genetic testing ...... 219 7.1.5 Yield of diagnostic investigations ...... 219 7.1.6 Commentary ...... 223 7.2 Implications of making a diagnosis on research imaging or genetic testing . 228 7.2.1 Commentary ...... 228 7.3 Health economics of diagnostic investigation ...... 230 7.3.1 Modelled diagnostic pathway ...... 231 7.3.2 Costing analysis ...... 239 7.3.3 Commentary ...... 244

Chapter 8: Discussion ...... 249

8.1 Introduction ...... 249 8.2 Study design ...... 250 8.3 SEI definition ...... 251 8.4 Epidemiology ...... 254 8.5 Aetiologies ...... 255 8.6 Clinical features ...... 257 8.7 Diagnostic investigation ...... 258 8.8 Significance of study findings ...... 260 8.9 Unanswered questions and future directions ...... 261

References ...... 263

Appendices ...... 319 A Phenotypes of the single gene causes of ‘severe’ infantile epilepsies ...... 321 B Study clinical assessment case report form ...... 343 C Known and candidate epilepsy genes included in the MIPS gene panels ...... 357 D Genes included in targeted analysis of whole exome sequencing ...... 359 xv

E Classification scheme for variants identified on whole exome sequencing ...... 377 F Criteria for approval of a waiver of consent for the ascertainment phase of this study ...... 379 G Human Research and Ethics Committee approval ...... 381 H Parent information and consent forms ...... 395 I Aetiologies of SEI and timing and method of diagnosis in 114 infants ...... 411 J Classification of seizure types in 114 infants using three versions of the ILAE seizure type classification ...... 417 K Epileptic syndrome classification in 114 infants ...... 419 L Examples of how prototypic and variant epileptic syndromes were assigned ...... 423 M Patient data and investigation costs used to model diagnostic pathways ...... 427

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List of Tables

Table 1.1 Structural (non-acquired) causes of infant epilepsy 36

Table 1.2 Metabolic disorders associated with infantile epilepsy 38

Table 1.3 Chromosomal abnormalities associated with infant epilepsy 40

Table 1.4 Single gene (non-malformative, non-metabolic) disorders causing infantile epilepsy 43

Table 1.5 Tiered investigations currently performed in infants with severe epilepsies of unknown cause 64

Table 1.6 Use of next generation sequencing in epilepsy 66

Table 1.7 Comparison of definitions of the terms used in the literature for ‘severe’ epilepsies 78

Table 1.8 Applicability of epilepsy prognostic factors to the infant population 81

Table 1.9 Studies looking at the relationship between seizure frequency and developmental outcome 83

Table 3.1 Potential criteria for a definition of ‘severe’ epilepsy in patients with well-described infantile epileptic syndromes 94

Table 3.2 Data used to determine aetiology and electroclinical phenotype 100

Table 3.3 Clinical and demographic data obtained from the medical record 101

Table 3.4 Definitions of epileptic syndromes used in this study 104

Table 3.5 Current standard investigations for aetiologic diagnosis of infants with SEI with unknown cause at time of presentation. 116

Table 3.6 Cost of pathology tests 118

Table 3.7 Costs of tiers of tests in the diagnostic pathway 119

Table 3.8 Costs of confirmatory testing of suspected diagnoses 123

Table 3.9 Simulated diagnostic pathways modelled in this economic evaluation assuming that infants progressed through the pathway until an aetiologic diagnosis was made 125

Table 3.10 Simulated diagnostic pathways modelled in this economic evaluation assuming that infants progressed through the pathway until an aetiologic diagnosis was made only if seizures were ongoing 125

Table 3.11 Study time frames 127

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Table 4.1 Number of EEGs performed on children less than two years old by site and year of EEG 132

Table 4.2 Number of NICU database entries listing ‘seizures’ by site and year of EEG 135

Table 4.3 Epileptic syndromes in infants with epilepsy not meeting clinical criteria for SEI 145

Table 4.4 Aetiologies in infants with epilepsy not meeting clinical criteria for SEI 145

Table 5.1 Aetiologies of SEI in 114 infants 152

Table 5.2 Aetiologies of SEI using a modified classification 154

Table 5.3 Genetic basis of malformative and metabolic aetiologies 157

Table 5.4 Suspected aetiologies in infants with unknown cause for SEI 159

Table 5.5 Incidence of SEI by aetiologic groups using a modified classification 166

Table 6.1 Seizure types present at onset and evolution of the epilepsy in 114 infants with SEI 174

Table 6.2 Epileptic syndromes at epilepsy onset 177

Table 6.3 Incidence of ILAE neonatal and infantile severe epileptic syndromes (and their variants) 180

Table 6.4 Aetiologies by epileptic syndrome at epilepsy onset 181

Table 6.5 Effect of the first antiepileptic agent used to treat spasms 185

Table 6.6 Epilepsy evolution and seizure outcome at two years old or death by epileptic syndrome at onset 189

Table 6.7 Epilepsy evolution and seizure outcome at two years old by aetiology 191

Table 6.8 Development by epileptic syndrome prior to epilepsy onset and at two years old 203

Table 6.9 Development by aetiology prior to seizure onset and at two years old 205

Table 7.1 Timing and method of SEI diagnosis in 114 infants 215

Table 7.2 Occult focal cortical dysplasia identified on research imaging review 218

Table 7.3 Clinical investigations and research imaging review in 86 infants with unknown cause at the time of presentation 220 xviii

Table 7.4 Yield of each tier of investigations in infants with unknown cause at the time of presentation 222

Table 7.5 Research genetic testing performed on infants with unknown aetiology after clinical investigation and research imaging review 223

Table 7.6 Number of diagnoses made using each diagnostic pathway modelled 232

Table 7.7 Number of investigations performed and diagnoses made at each step if all 86 infants with unknown cause at the time of presentation are investigated until aetiology identified or reach end of diagnostic pathway 233

Table 7.8 Number of investigations performed and diagnoses made at each step if all 86 infants with unknown cause at the time of presentation are investigated until aetiology identified only if seizures are ongoing 234

Table 7.9 Cost and yield of diagnostic pathways if all 86 infants with unknown cause at the time of presentation are investigated until aetiology identified or reach end of diagnostic pathway 240

Table 7.10 Additional and saved costs per scenario if all 86 infants with unknown cause at the time of presentation are investigated until aetiology identified or reach end of diagnostic pathway 241

Table 7.11 Cost and yield of diagnostic pathways if all 86 infants with unknown cause at the time of presentation are investigated until aetiology identified only if seizures are ongoing 242

Table 7.12 Additional and saved costs per scenario if all 86 infants with unknown cause at the time of presentation are investigated until aetiology identified only if seizures are ongoing 243

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List of Figures

Figure 1.1 Important EEG patterns in infants 12

Figure 1.2 Genetic aetiologies of the ‘severe’ infant epileptic syndromes 58

Figure 3.1 Study design diagram 90

Figure 3.2 Study screening sites 98

Figure 4.1 Flow diagram showing the process of identifying infants with SEI from EEG reports 134

Figure 4.2 Flow diagram showing the process of identifying infants with SEI from NICU database entries 137

Figure 5.1 Examples of malformations of cortical development in infants in this study. 155

Figure 6.1 Age of epilepsy onset in 114 infants with SEI 173

Figure 6.2 Seizure outcome at two years old by epileptic syndrome at onset 188

Figure 7.1 Diagnostic pathway without whole exome sequencing 235

Figure 7.2 Diagnostic pathway with whole exome sequencing 236

Figure 7.3 Diagnostic pathway without whole exome sequencing (version 2) 237

Figure 7.4 Diagnostic pathway with whole exome sequencing (version 2) 238

Abbreviations

ACTH Adrenocorticotropic hormone AED Antiepileptic drug ASD Autism spectrum disorder BFIE Benign familial infantile epilepsy BFNIE Benign familial neonatal/infantile epilepsy BFNE Benign familial neonatal epilepsy BMEI Benign myoclonic epilepsy of infancy CMA Chromosomal microarray CNV Copy number variant CSF Cerebrospinal fluid CT Computerised tomography scan DNA Deoxyribonucleic acid DQ Developmental quotient EE Epileptic encephalopathy EEG Electroencephalogram EIEE Early infantile epileptic encephalopathy (Ohtahara syndrome) EIMFS Epilepsy of infancy with migrating focal seizures EME Early myoclonic encephalopathy FCD Focal cortical dysplasia FS Febrile seizures GEFS+ Genetic epilepsy with febrile seizures plus GGE Genetic generalised epilepsy GLUT1 Glucose transporter 1 GP General practitioner HIE Hypoxic-ischaemic encephalopathy IED Interictal epileptiform discharge ILAE International League Against Epilepsy IS Infantile spasms IQ Intelligence quotient IQR Interquartile range LGS Lennox-Gastaut syndrome MAE Epilepsy with myoclonic-atonic seizures MCHN Maternal and child health nurse MCRI Murdoch Childrens Research Institute MENPD Myoclonic encephalopathy in a non-progressive disorder MHW Mercy Hospital for Women MIPS Molecular inversion probes MMC Monash Health (formerly Monash Medical Centre) MRI Magnetic resonance imaging MRS Magnetic resonance spectroscopy mTOR Mammalian target of Rapamycin NGS Next generation sequencing NICU Neonatal intensive care unit PCR Polymerase chain reaction

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PET Positron emission tomography PICF Parent information and consent form PMG Polymicrogyria RCH Royal Children’s Hospital Melbourne RWH Royal Women’s Hospital SEI Severe epilepsies of infancy SFE Structural focal epilepsy / symptomatic focal epilepsy SUDEP Sudden unexpected death in epilepsy T Tesla TS Tuberous sclerosis UKISS United Kingdom Infantile Spasms Study VABS Vineland adaptive behaviour scale VCGS Victorian Clinical Genetics Service VOUS Variant of unknown significance WES Whole exome sequencing WGS Whole genome sequencing

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Chapter 1: Literature review

1.1 Introduction

This chapter is a critical review of the literature of infantile epilepsy, with a focus on epidemiology, clinical features and aetiologies.

The chapter begins with a series of definitions to orientate the reader to the group of patients considered to have an infantile epilepsy. This is followed by a discussion of the neurobiology of the infant brain as it relates to epilepsy. The next section considers the clinical features, both epileptic and non-epileptic seen in these conditions. Following that are sections that consider the aetiologies of infantile epilepsies with a focus on genetic causes, how the aetiology is investigated, and the health economics of diagnostic investigation. A discussion of the epidemiology of infantile epilepsy concludes the analysis of all infant epilepsy. The final section of this chapter considers the question, ‘what is severe epilepsy of infancy’?

1.2 Description of the infantile epilepsies

Infantile epilepsies are a group of conditions with the common symptom of recurrent epileptic seizures, defined in this study as occurring in the first 18 months of life. The infantile epilepsies are a subset of all seizure disorders, and paroxysmal neurologic disorders in this age group.

The most widely accepted definition of an epileptic seizure is that proposed by the International league against epilepsy (ILAE) in 2005: ‘An epileptic seizure is a transient occurrence of signs and/or symptoms due to the abnormal excessive or synchronous neuronal activity in the brain’(Fisher et al., 2005).

The ILAE also states: ‘Epilepsy is a disorder of the brain characterised by an enduring predisposition to generate epileptic seizures and by the neurobiologic, cognitive, psychological and social consequences of this condition’(Fisher et al., 2005) . This definition requires two unprovoked seizures more than 24 hours apart, a single seizure with a greater than 60% chance of further seizures in the next two years, or diagnosis of an epileptic syndrome (Fisher et al., 2014). In most instances, epilepsy is practically defined by paediatric clinicians as at least two unprovoked seizures over a greater than 24 hour period, typically excluding neonatal seizures. It is important to recognise that,

although not explicitly stated in this definition, epilepsy is in fact not a single disorder, but rather a group of conditions due to a variety of causes, with the shared symptom of recurrent seizures. The term ‘epilepsies’ is often preferred to ‘epilepsy’ to represent this heterogeneity.

A variety of definitions of infancy exist, most based on age, but some based on developmental stage (pre-walking). The World Health Organisation considers infancy to span the ages of 1-12 months (http://www.who.int/hiv/pub/guidelines/arv2013/intro/keyterms/en/), in line with the most commonly used definitions in medical and sociologic literature. However, the age range applied to infancy varies in the medical literature, some defining this period as broadly as birth-2 years (Johnson & Blasco, 1997). With respect to epilepsy, a definition of infancy should consider biologic age-groupings as well as taxonomic norms. For example, some age-related epileptic syndromes of ‘infancy’ can begin outside the 1-12 month age period. However, the epileptic syndromes of infancy rarely have onset after 18 months of age; Dravet syndrome begins before 15 months, and just 1% of infantile spasms have onset after 18 months old (Lombroso, 1983; Scheffer, 2012; Yamatogi & Ohtahara, 2002). Further, while the ‘early childhood’ epileptic syndromes including Epilepsy with myoclonic-atonic seizures (MAE) and Lennox- Gastaut syndrome (LGS) can occasionally begin before 18 months old (Bureau, Thomas, & Genton; Yamatogi & Ohtahara, 2002), the peak age onset of those syndromes is considerably later, at 3-4 years and 3-5 years respectively (Bureau et al.; P. R. Camfield, 2011; Stephani, 2006; Yamatogi & Ohtahara, 2002). As such, in this study, infancy is defined as 0-18 months old.

The neonatal period is typically defined as the first 28 days after birth (http://www.who.int/topics/infant_newborn/en/epe ). Some definitions consider the neonatal period to be separate from infancy, others (as above, and including this study) consider it a subgroup of the infantile period.

As in other age groups, single seizures and provoked or ‘acute symptomatic’ seizures in infancy are generally not considered epilepsy, although the 2014 definition indicates that a single seizure with a high recurrence risk may be defined as epilepsy (Fisher et

al., 2014). Further, in earlier classification documents, some non-provoked recurrent neonatal seizures were not afforded a diagnosis of epilepsy. However, in the most recent classification system, neonatal seizures are no longer regarded separately, and are now considered true epilepsies (Berg et al., 2010; ILAE, 1989).

The most pertinent additional example in infancy is that of febrile seizures (FS), the most common seizure disorder in this age group (rev in (P. Camfield & Camfield, 2015a)). While some consider FS to be a ‘benign epilepsy’ in infants and young children, it is not considered epilepsy in standard definitions and classifications. There are many reasons for this, including the consideration of fever as a provoking factor, and single or rare seizures only in many infants. Additionally, FS occur frequently, occurring in approximately 3% of the Caucasian population and reported at a higher rate in populations in Japan, Finland and Guam (P. Camfield & Camfield, 2015a; Verity, Butler, & Golding, 1985a). Morbidity is rare and only a small percentage go on to have later epilepsy (Nelson & Ellenberg, 1976; Verity, Butler, & Golding, 1985b). Thus, it is considered to be distinct from epilepsy, and further discussion of infantile epilepsies in this chapter will not include this group.

Paroxysmal disorders are frequent in infancy, with incidences estimated at 8.9-12.9%, and 25%, in the first year and first two years of life respectively (Reerink et al., 1995; Visser et al., 2010). In a population-based prospective cohort study in which parents were asked about paroxysmal episodes that had occurred in their child, epileptic seizures made up only a minority of paroxysmal events; approximately 90% were felt to be non-epileptic in nature (Visser et al., 2010). While most non-epileptic paroxysmal disorders occur in infants without epilepsy, a mix of epileptic and non-epileptic paroxysmal events may be seen in some, particularly those with severe disorders.

A broad range of non-epileptic episodes is recognised. While no standardised classification system exists, non-epileptic episodes can be divided broadly into episodes of altered awareness, altered movement, or altered respiration. Episodes can be normal or exaggerated physiologic variants such as sleep myoclonus and apnoea of prematurity, relatively ‘benign’ episodes such as breath-holding spells and migraine variants, and

potentially sinister episodes such as cardiogenic syncope and opsoclonus (rev in (Paolicchi, 2002; Park, Lee, Lee, Lee, & Lee, 2015)).

Positive diagnoses for some non-epileptic episodes can be made solely from description or observation of the episode and the context in which it occurs. However, some are misdiagnosed as epilepsy. Paroxysmal episodes in neonates are an important example. Here, many episodes clinically diagnosed as seizures are shown to not have an epileptic basis when captured on EEG (Malone et al., 2009; Mizrahi & Kellaway, 1987) . Even in older children, rates of misdiagnosis of epilepsy are high, ranging from 5-23% in previous studies (Leach, Lauder, Nicolson, & Smith, 2005; Stroink et al., 2003). Where the nature of the episode is unclear, it is sometimes necessary to exclude an epileptic basis by video-EEG monitoring. At the individual patient level, erroneous diagnoses of epilepsy may result in administration of, and side effects due to, unnecessary treatments, anxiety related to the ‘diagnosis’ and its ‘prognosis’, and failure to appropriately diagnose and treat the actual aetiology. At a population level, misdiagnoses impact on the quality of clinical research and the accuracy of epidemiologic studies of epilepsy.

1.3 Neurobiology of infantile epilepsies

Epilepsy, crudely considered a disorder of neuronal excitability, can arise as a consequence of a wide range of perturbations of brain development and neuronal function. Many factors contribute to the balance of excitation and inhibition in the brain, both at the individual neuron and neuronal network level.

At a gross anatomical level, the bulk of brain development occurs in the prenatal and early postnatal periods. Many processes including generation of neurons, cellular proliferation and apoptosis and neuronal migration are required for normal prenatal development. Major postnatal aspects of brain development include refinement of synaptic connections, completion of myelination and the formation of neuronal networks. These processes are tightly controlled by a complex array of factors, including regulation of gene transcription, intra- and intercellular signalling, and physical factors such as location of neighbouring neurons and non-neuronal support structures (rev in (Stiles & Jernigan, 2010)).

Many types of neurons are present, the two main types being pyramidal neurons (excitatory) and interneurons (inhibitory). The actions of these neurons depends on their generation of action potentials via depolarisation of the neuronal membrane. Action potentials are generated by net positive ion fluxes into the neuron, occurring via voltage-gated ion channels, changes in extracellular ion concentration or the actions of neurotransmitters on ligand-gated ion channels. The generation of an action potential leads to release of a neurotransmitter from the pre-synaptic membrane of that neuron, where it acts on ligand-gated channels in the post-synaptic terminal of neuron(s) to which it is connected. Pyramidal neurons release the excitatory neurotransmitter glutamate which acts on AMPA and NMDA receptors, and interneurons the (usually) inhibitory neurotransmitter GABA which acts on GABA receptors (Kandel, 2013).

The actions of neurotransmitters vary across development, with changes in synaptic and extrasynaptic actions as development advances, and indeed variability in the cellular effects exerted by particular neurotransmitters. A good example of the latter is the effect of GABA (rev in(Ben-Ari, 2014)). Unlike in mature neurons, where the effect of GABA is to hyperpolarise the cell, the opposite occurs in immature neurons in the first few weeks of life (Ben-Ari, Cherubini, Corradetti, & Gaiarsa, 1989; Obata, Oide, & Tanaka, 1978). This difference is due to expression of different cellular chloride-cation transporters as the intracellular chloride level is a factor in determining the electrical response to GABA (rev in (Kahle et al., 2008)). In the mature neuron, the major chloride-cation transporter, KCC2 couples chloride transport to the flow of potassium ions, resulting in a net outward flux of chloride and lowering the intracellular chloride concentration. Upon activation of the GABA receptor, there is a passive flow of chloride ions into the cell through the ligand-gated chloride channel, and results in hyperpolarization of the neuron. In the first few weeks of postnatal development, the main chloride channel is NKCC1. This channel couples the flow of chloride ions to the flow of sodium ions, resulting in a net flow of chloride into the cell, raising the intracellular chloride concentration. Upon activation of the GABA receptor, chloride moves out of the cell, and causes the cell to depolarise (rev in (Ben-Ari, 2002; Gamba, 2005)). Thus, in the neonatal period, GABA can actually be excitatory. This phenomenon appears to be required for many aspects of normal brain development as genetic or pharmacologic disruption of the NKCC1 channel leads to abnormalities in

neuronal number, maturation, structure, function and networks (Cancedda, Fiumelli, Chen, & Poo, 2007; Reynolds et al., 2008). Excitatory actions of GABA are strongly correlated in vitro with the susceptibility of a cell to develop seizures (Dzhala & Staley, 2003). This is likely an important contributor to the higher rate of seizures in neonates than at older ages, and lack of effect (or even paradoxical actions) of some AEDs (Connell, Oozeer, de Vries, Dubowitz, & Dubowitz, 1989; Scher, Alvin, Gaus, Minnigh, & Painter, 2003).

Pathologic processes that produce seizures are numerous; two important examples are mentioned here. Correct spatial distribution of neuron types is important, as evidenced by disorders of interneuron migration such as in ARX mutations (Price et al., 2009; Stromme et al., 2002). Further, across development, the relative expression of different genes changes, as the particular proteins required for normal neuronal function changes with age. The age of onset of some genetic epilepsies, including those due to mutations in KCNQ2, SCN2A, SCN8A and GRIN2A, is tightly correlated with temporal expression of these genes (Carvill, Regan, et al., 2013; Howell et al., 2015; Kanaumi et al., 2008; Larsen et al., 2015; Lemke et al., 2013; Lesca et al., 2013; Oliva, Berkovic, & Petrou, 2012; Weckhuysen et al., 2012).

Abnormal development of cognitive, language and motor function often coexists with infant-onset epilepsies (Chevrie & Aicardi, 1978; Czochanska, Langner-Tyszka, Losiowski, & Schmidt-Sidor, 1994; Matsumoto et al., 1983). A number of neurobiologic factors contribute to this. Abnormal development can be due to an underlying aetiology that results in both epilepsy and delayed development. Additionally, alterations in developmental trajectory can occur as a result of seizures and interictal epileptiform discharges (IEDs). The cellular and network mechanisms by which this occurs are not well understood. But, seizures can produce both functional and structural changes in the developing brain, including changes in cellular excitability, neuronal connections, neurogenesis and cellular survival. It is notable that function and survival of neurons, and development and function of neuronal networks, is activity-dependent. Aberrant neuronal activity (whether increased or decreased), such as that of seizures and IEDs, thus impacts on neuronal networks and ultimately development (rev in (Brooks-Kayal, 2011)).

1.4 Clinical features of infant epilepsies

The infantile epilepsies are a heterogeneous group of conditions, with a variety of epileptic and non-epileptic clinical features and comorbidities.

1.4.1 Epileptic features Seizure types and semiology

Seizures can be described at two levels – semiologic description, and seizure type. The former is purely a description of the clinical features of the event (e.g. version, tonic stiffening, clonic jerking etc.), the latter classically incorporates both semiology and ictal EEG (e.g. myoclonic, tonic, focal etc.). Seizure types are grouped into ‘focal’, ‘generalised’ or unknown based on whether the seizure arises in unilateral networks, originates in and rapidly involves bilateral networks, or whether this is unclear (Berg et al., 2010; Engel & International League Against, 2001; ILAE, 1981, 1989)

There are a number of features of seizure semiology and seizure type that are important or peculiar to infancy, and limitations of the ILAE classification of seizure types in this age group, which are considered here.

The ILAE classification assumes strong correlation between seizure semiology and EEG findings. This assumption holds well for seizures in more mature brains, but is frequently incorrect for infants. In particular, tonic posturing (bilateral or unilateral) and myoclonic jerks can occur in either focal or generalized seizures (M. S. Duchowny, 1987; Hamer, Wyllie, Luders, Kotagal, & Acharya, 1999; Nordli, Bazil, Scheuer, & Pedley, 1997). These features mean that, in infants, classification of seizure type relies not only on semiology, but also heavily on additional supporting information, including EEG patterns, other seizure types, age and developmental history(Nordli et al., 1997).

While a range of seizure types occurs in infants, the spectrum of seizure types is different to that of older ages. Some seizure types, e.g. epileptic spasms, are seen predominantly in infancy (Lombroso, 1983). Other types, e.g. primarily generalized tonic-clonic seizures are not seen in infancy (Hamer et al., 1999; Nordli et al., 1997). A study of seizures in patients with refractory seizures during the first three years of life

reported that four seizures types accounted for over 80% of seizures: infantile spasms, tonic seizures, clonic seizure and “hypomotor” seizures (Hamer et al., 1999).

Although epileptic spasms are a common seizure type in infants, they have proved difficult to classify as there is debate as to whether they are a focal or generalized seizure type, or whether both are possible. The arguments for each depends partly on whether the classification is based on mechanistic networks (spasms usually generalized) or aetiology (spasms may be focal). Their listing in the classification has changed with time – initially listed as ‘unclassified’ (ILAE, 1981), then of ‘unknown’ onset (Berg et al., 2010), and most recently spasms can be classified as a focal, generalized or unknown seizure type to account for all possibilities (http://www.ilae.org/visitors/centre/Class-Seizure.cfm).

Some semiologic differences are noted in seizures occurring in the infant brain compared with those in children and adults, particularly with respect to focal seizures. Reported differences (M. S. Duchowny, 1987; Hamer et al., 1999; Luna, Dulac, & Plouin, 1989; Wyllie et al., 1993; N. Yamamoto et al., 1987) include:

 Seizures with prominent automatisms are less common in infancy. Where automatisms occur in infancy, they tend to be simple, are usually oral, and may be more difficult to distinguish from normal behaviour.  Focal dystonic posturing of limbs is rarely seen in infants.  Symmetric tonic posturing is more common during infancy.

Further, the significance of observed ictal behaviours may be more difficult to determine. Examples (Brockhaus & Elger, 1995; M. S. Duchowny, 1987; Holmes, 1986; Luna et al., 1989) include:

 Difficulty in determining whether an alteration in responsiveness represents altered consciousness versus sensory preoccupation versus a motor impairment that prevents a response. In the 2010 classification, the removal of the ‘simple partial seizure versus complex partial seizure’ distinction present in the 1989 ILAE classification resolved this difficulty (Berg et al., 2010; ILAE, 1989).

 Apparent absence of an aura. This may simply reflect the fact that an infant is not be able to describe an aura (as opposed to sensory symptoms not being present), as subtle behavioural changes that precede focal seizures in infants are common.

Such differences between infants and older children and adults are felt to reflect the immaturity of the infant brain, both with respect to differences in connectivity altering pathways and networks through which seizures arise and spread, as well as differences in ability to describe or convey the experience of the seizure.

EEG features

EEG is performed in infants to investigate interictal and ictal patterns that support or confirm the clinical impression of seizure type, epileptic syndrome, epilepsy severity (e.g. presence of encephalopathic features, frequency of seizures) and, rarely, specific aetiologic diagnosis The normal infantile EEG varies significantly with post-conceptual age, and state of awareness, such as wake or sleep. Thus, interpretation of any infant EEG must be done with knowledge of these factors (Peter Kellaway, Petersén, & World Federation of Neurology., 1964; Niedermeyer, Schomer, Lopes da Silva, & Ovid Technologies Inc., 2011).

The interictal EEG of an infant with epilepsy may be normal or abnormal. Abnormalities can be focal, multifocal or diffuse. There is not a straightforward correlation between the finding of focal features and a focal aetiology, nor do generalized features correlate with a diffuse cause (Bureau et al.; Chugani et al., 1990; E. K. Gaily, Shewmon, Chugani, & Curran, 1995). Abnormal findings may include IEDs and disturbances of background activity. Frequently seen abnormalities include spike or sharp waves, excessive slowing, absences of physiologic rhythms and presence of encephalopathic patterns. Well-described encephalopathic patterns (Figure 1.1) include:

 burst-suppression, a periodic pattern characterized by bursts of often irregular high voltage paroxysmal activity interspersed with periods of flattening of the EEG that is associated with EIEE and EME (as well as preterm gestation,

highlighting the importance of knowing gestational age in interpreting EEG findings) (Niedermeyer et al., 2011)  hypsarrhythmia, characterised by chaotic irregular high-amplitude slow waves interspersed with multifocal asynchronous spike and sharp waves, without normal background activity, classically seen in West syndrome (in fact, modified hypsarrhythmia, indicating some preservation of background activity, synchronization of spike-wave activity or localizing features, is seen more commonly in West syndrome than classical hypsarrhythmia) (Hrachovy, Frost, & Kellaway, 1984).

Sleep recordings can be critical in detecting some encephalopathic patterns, as the burst- suppression of EME may be confined to sleep (Ohtahara & Yamatogi, 2003), as occasionally may hypsarrhythmia in West syndrome (Jeavons & Bower, 1961). Serial EEG may also be of value in some infants with ongoing or evolving seizures, as interictal abnormalities may evolve or resolve over time (in infants with a previously normal EEG, or a previously abnormal EEG showing different patterns) (Coppola, Plouin, Chiron, Robain, & Dulac, 1995; Yamatogi & Ohtahara, 2002).

Figure 1.1 Important EEG patterns in infants

A) burst-suppression and B) hypsarrhythmia EEG patterns

As seizure frequency is often high in infants, clinical or electrographic seizures may be recorded, even during a brief routine EEG lasting 30 minutes. Seizures can have an apparently ‘generalised’ ictal onset, such as the electrodecrements or high amplitude slow wave with admixed fast activity seen in tonic seizures or spasms (P. Kellaway, Hrachovy, Frost, & Zion, 1979). Ictal rhythms can also have a focal onset, in any

cortical location. A wide range of focal ictal rhythms can be seen, sometimes within the one patient, including rhythmic delta, theta or alpha, low voltage fast activity, trains of spikes or attenuation (Nordli, Kuroda, & Hirsch, 2001). Focal ictal rhythms can spread from onset location to adjacent or contralateral regions, but true secondary generalisation is not common (M. S. Duchowny, 1987). In contrast, intra-ictal activation (‘migration’) of a focus in the contralateral hemisphere may be seen (Coppola et al., 1995). Clinical or electrographic seizures in infants may cluster, occur in a periodic fashion or become continuous.

Infantile epileptic syndromes

This section considers epileptic syndromes, which are recognizable groups of clinical and electroclinical characteristics. A number of well-described epileptic syndromes of infancy exist, including syndromes considered ‘benign’ due to favourable seizure and developmental outcome, and more severe syndromes with high likelihood of poor outcomes. Each is described below. Many infants have epilepsies that do not fit a well- described syndrome; these ‘complex’ phenotypes are also considered here. This section does not consider the aetiologies associated with each epileptic syndrome; this follows later in this Chapter.

Benign familial neonatal and infantile epilepsies: ‘Benign’ familial neonatal seizures were first reported by Rett and Teubel in 1964 (Bureau et al.). Now, three epileptic syndromes with focal seizures and good outcome in the neonatal and infant period are recognised: BFNE, BFNIE and BFIE (Kaplan & Lacey, 1983; Vigevano et al., 1992). An autosomal dominant family history is often present (although may not be recalled), but sporadic cases with a similar electroclinical picture can occur. The main factor which differentiates these clinically during infancy is the mean age of seizure onset within a family, being less than 1 month, 1-4 months or greater than 4 months respectively. The epilepsy phenotype is similar for all three syndromes (Deprez, Jansen, & De Jonghe, 2009). Seizures often occur in clusters and consist of brief, focal motor manifestations (usually version) with subsequent tonic and/or clonic components and autonomic features such as apnoea. The interictal EEG is typically normal. Seizures are usually treatment-responsive. These conditions have been considered ‘benign’ as seizures are usually treatment-responsive, developmental outcomes are normal, and

spontaneous seizure remission occurs within the first year of life in most. However, treatment-refractory seizures, and abnormal developmental outcomes are reported in rare familial cases (Borgatti et al., 2004; Dedek, Fusco, Teloy, & Steinlein, 2003); the term ‘self-limited’ is now preferred. Some clinical variability is reported after the infantile period, with seizures after infancy in some patients (Grinton et al., 2015), and childhood-onset paroxysmal kinesogenic dystonia or choreoathetosis (a constellation known as infantile convulsions and choreoathetosis) in some with BFIS or their relatives (Szepetowski et al., 1997).

Benign myoclonic epilepsy of infancy: Benign myoclonic epilepsy of infancy (BMEI) is an extremely rare epileptic syndrome with onset between four months and three years of age in developmentally normal children, first described by Dravet and Bureau in 1981(Dravet & Bureau, 1981). It is conceptualized as an infantile form of idiopathic (genetic) generalized epilepsy. The cause is unknown, although a 2:1 male predominance, and a family history of epilepsy (other epileptic syndromes, there are no reported familial cases of BMEI) in one-third suggest a genetic basis. Multiple-daily myoclonic seizures occur in most, and approximately 20% also have FS. The interictal EEG is typically normal. Seizures are controlled with valproate monotherapy in approximately 80%, and with one add-on medication in most of the remaining patients. The seizure outcome is typically good, with remission within a year of onset in most (Auvin et al., 2006). Later recurrence of seizures occurs in a minority (Mangano et al., 2011; Moutaouakil, El Otmani, Fadel, El Moutawakkil, & Slassi, 2010). The consideration of this syndrome as ‘benign’ has been questioned following reports of some degree of learning and behavioural difficulties, or intellectual disability in 10-81% of patients at follow-up (Lin et al., 1998; Mangano et al., 2011; Zuberi & O'Regan, 2006).

Early infantile epileptic encephalopathy (Ohtahara syndrome): EIEE, also known as Ohtahara syndrome, was first described in 1976 (Ohtahara & Yamatogi, 2003; Yamatogi & Ohtahara, 1981, 2002). This syndrome has onset typically in the neonatal period (with fetal seizures suspected in some cases), although it may begin as late as three months of age (Clarke, Gill, Noronha, & McKinlay, 1987).. The clinical hallmarks are frequent, predominantly tonic seizures, and a markedly abnormal EEG showing

burst-suppression patterns. Seizures are typically refractory to treatment, except where an operable lesion is identified as the cause of the EIEE. Seizure control with vigabatrin or zonisamide is rarely reported (P. S. Baxter et al., 1995; Ohno, Shimotsuji, Abe, Shimada, & Tamiya, 2000). Seizure remission is reported in some patients after the infantile period, particularly with some genetic causes (e.g. KCNQ2; differences between individual causes are discussed further in the aetiologies section), but persistent seizures are seen in many (Weckhuysen et al., 2012; Yamatogi & Ohtahara, 2002). EIEE is considered the earliest of the age-dependent severe epileptic syndromes. Evolution to other age-dependent syndromes is frequently seen, with evolution to West syndrome reported in 75% and LGS in 12% (Yamatogi & Ohtahara, 1981, 2002), with focal and multifocal epilepsies also reported. The outcome of EIEE is poor in most patients, and death frequently occurs in infancy or childhood. Severe or profound cognitive impairment is seen in most survivors, including in some genetic epilepsies with early resolution of seizures, although the developmental outcome may be relatively better in the case of prompt surgical treatment of operable structural abnormalities (Komaki et al., 1999; Pedespan et al., 1995; Weckhuysen et al., 2012).

Early myoclonic encephalopathy: EME was described by Aicardi and Goutières in 1978, and consists of early onset (within the first month of life in 96%) of predominantly myoclonic seizures, with a burst-suppression EEG that is more prominent in sleep (may be solely in sleep) (Aicardi & Goutieres, 1978) (Murakami, Ohtsuka, & Ohtahara, 1993) . Seizures are typically refractory to AEDs, although seizure freedom may follow institution of specific therapies in some treatable metabolic conditions (P. Baxter, 2001; Hunt, Stokes, Mc, & Stroud, 1954). Additional seizure types are commonly seen, including tonic seizures (in approximately three-quarters, often with onset some months after the myoclonic seizures) (Djukic, Lado, Shinnar, & Moshe, 2006). Age-dependent evolution of the epileptic syndrome is not common, and the burst-suppression EEG can persist throughout childhood (Dalla Bernardina et al., 1983). However, some patients display a brief transition to West syndrome, before the burst-suppression EEG reappears. Approximately 50% of patients die in childhood, usually in the first year of life. The developmental outcome is very poor in most survivors (Aicardi & Goutieres, 1978; Dalla Bernardina et al., 1983).

It is sometimes difficult to distinguish EME from EIEE, as both myoclonic and tonic seizures can occur in each condition, and the burst-suppression EEG pattern is common to both. Debate exists as to whether the conditions are distinct, or whether they represent a continuum (Djukic et al., 2006).

Epilepsy of infancy with migrating focal seizures: The syndrome of EIMFS was first reported in 1995 (Coppola et al., 1995). The original diagnostic criteria described multifocal seizures without identifiable cause, onset in the first six months of life, with the characteristic ictal EEG pattern of seizures arising independently in both hemispheres, migrating from one hemisphere to the other. Seizures are intractable, developmental plateauing or regression is seen and severe cognitive impairment ensues.

Subsequent reports have further delineated the epilepsy, with three distinct phases now recognized (Coppola, 2009). The ‘early phase’ begins between one day and six months of age and lasts weeks to months. During this phase, seizures are relatively infrequent, recurring at weekly-monthly intervals, and the interictal EEG is normal in up to 30% of cases. The ‘stormy phase’ begins between one and 12 months of age, consisting of extremely frequent (often multiple per hour, and sometimes continuous) seizures that often occur in clusters. During this phase, the interictal EEG has a slow background and there are multifocal IEDs. The ‘burnt out phase’ typically starts between one and five years old. Here, seizures are relatively infrequent, with clusters of seizures or episodes of status epilepticus typically occurring predominantly in the context of intercurrent illness. Transition to other age-dependent epileptic syndromes is uncommon, and infantile spasms are reported in only 7% (Coppola et al., 1995).

Other non-epileptic features of this condition include acquired microcephaly and hypotonia evolving to a spastic quadriparesis. Movement disorders, severe gut dysmotility and evolving brain imaging abnormalities (diffuse atrophy, white matter abnormalities) are also frequently seen (Coppola, 2009; McTague et al., 2013).

Infantile spasms/West syndrome:

Infantile spasms were first described in 1841 by Dr James West, who described the seizures he witnessed in his son (Duncan, 2001). West syndrome is typically considered

the triad of epileptic spasms with onset in infancy, EEG showing hypsarrhythmia (although modified hypsarrhythmia may also be seen), and developmental arrest or regression (Bureau et al.). West syndrome is the most common of the ‘severe’ infant epileptic syndromes (Cowan & Hudson, 1991)

Over time in the literature, the terms infantile spasms and West syndrome have been used somewhat interchangeably, this variable use in part reflecting that hypsarrhythmia and abnormal development are not always present. In 2004, a consensus statement for case definitions of infantile spasms and West syndrome was proposed, aimed at standardizing patient populations in clinical trials to facilitate comparisons between studies (Lux & Osborne, 2004). This report proposed that infantile spasms be considered an epileptic syndrome that occurs almost always before two years old (predominantly under one year), consisting of epileptic spasms occurring in clusters or singly, and usually but not universally associated with hypsarrhythmia and an abnormal developmental trajectory. It suggested West syndrome be considered a subgroup of infantile spasms, defined as epileptic spasms occurring in clusters, and associated with hypsarrhythmia; other authors have suggested similar, noting that infantile spasms without hypsarrhythmia should be considered a variant of West syndrome rather than a distinct syndrome (R. H. Caraballo et al., 2011). Developmental delay or regression was not a requirement in this proposed definition. Many of the studies reported below use the term infantile spasms (albeit with some variability in definitions and inclusion criteria); this term rather than West syndrome will be used for the remainder of this section.

Onset of infantile spasms is within the first year of life in 90-97% of patients (Chevrie & Aicardi, 1971; Lombroso, 1983; Pellock et al., 2010) Epileptic spasms are the first seizure type in many patients, but antecedent or concurrent seizures (e.g. focal seizures) or prior epileptic syndromes (e.g. EIEE) may occur (Donat & Lo, 1994; Yamatogi & Ohtahara, 1981).

Treatment of infantile spasms is pursued aggressively despite the observation that spasms typically resolve spontaneously with age (Riikonen, 1982). The goals of treatment are cessation of spasms and resolution of hypsarrhythmia, since both are

considered to adversely impact development. IS is one of the few infant epilepsies for which evidenced-based treatment is available, with corticosteroids and vigabatrin both extensively studied and widely used in clinical practice (Go et al., 2012). Both have been shown in a recent study to be more effective than ‘non-standard’ AEDs (Knupp et al., 2016). There has long been debate about superiority of the main corticosteroids used, intramuscular adrenocorticotrophic hormone (ACTH) and oral prednisolone (Go et al., 2012); a recent single-blind randomised control trial reported that ACTH was not superior to prednisolone (Wanigasinghe, Arambepola, Sri Ranganathan, Sumanasena, & Attanapola, 2015). A study of vigabatrin in drug-resistant IS showed resolution of spasms in 38% of infants treated, including a particularly high response rate in patients with tuberous sclerosis (TS), for which it has become first-line treatment (Chiron et al., 1990). The United Kingdom Infantile Spasms study (UKISS) compared the efficacy of these two treatments as first-line treatment, showing a higher rate of spasm cessation with steroids than vigabatrin (Lux et al., 2004). Additionally, developmental outcome was clearly superior with steroids than vigabatrin in infants with IS of unknown aetiology, with a 33 point higher (96 vs 63) median score on the Vineland Adaptive Behavioural Scale (VABS) at four years old (O'Callaghan et al., 2011). More recently, the International Collaborative Infantile Spasms Study showed that combination therapy with steroids and vigabatrin is more effective than steroids alone in stopping spasms, with 72% treated with combination therapy and 57% of patients on steroids alone free of spasms between days 14 and 42; developmental outcome is still under study (O'Callaghan et al., 2017).

Epileptic spasms are considered to be largely an age-dependent seizure type, with spasms ceasing in most by 3-5 years (Riikonen, 1982), resolution occurring in 75% by 12-14 months of age (Lux et al., 2005) and 87% at four years old (Darke et al., 2010). Spontaneous resolution without treatment can be seen (Hrachovy, Glaze, & Frost, 1991). Other seizure types occur in 49-78% (Darke et al., 2010; Koo, Hwang, & Logan, 1993; Lagae et al., 2010; Mohamed, Scott, Desai, Gutta, & Patil, 2011), and evolution to LGS is reported in 15-50% (Hrachovy & Frost, 2013; Trevathan, Murphy, & Yeargin-Allsopp, 1999; Yamatogi & Ohtahara, 2002)

Developmental delays prior to the onset of IS are seen in 68-85% of patients (P. Kellaway, Crawley, & Kagawa, 1959; Matsumoto et al., 1981; Riikonen, 1984). Plateau or regression is seen with or prior to the onset of IS in most patients, typically accompanied by a reduction in visual attention, loss of head control and hypotonia (Aicardi & Chevrie, 1978). While development may improve after the resolution of spasms, the overall outcome is poor in most. A review of multiple studies of patients with IS followed for an average of 31 months showed normal developmental outcome in only 16% (Hrachovy & Frost, 2003). Autism spectrum disorders are reported in one- third (Saemundsen, Ludvigsson, & Rafnsson, 2007). Predictors of better outcome include unknown aetiology, older age at IS onset (>4 months), absence of other seizure types prior to spasms, prompt treatment, prompt response to treatment and sustained treatment response (Eisermann et al., 2003; Hrachovy & Frost, 2003; Kivity et al., 2004; Koo et al., 1993; Lagae et al., 2010). A drop in intelligence quotient (IQ) of 16 points in those who had a delay of two months to treatment compared with those treated within a week of spasm onset highlights the importance of minimizing the duration of exposure to spasms and hypsarrhythmia (O'Callaghan et al., 2011).

Dravet syndrome: Charlotte Dravet first described Dravet syndrome in 1978. Previously known as severe myoclonic epilepsy of infancy (SMEI), the term Dravet syndrome is preferred as not all patients have myoclonus, particularly early in the condition, and with more awareness that carbamazepine is contraindicated (Scheffer, 2012).

Seizures begin at about six months of age, although rarely as late as 15 months (Depienne, Trouillard, et al., 2009), in a previously normal child, usually in the setting of a febrile illness or following vaccination (Berkovic et al., 2006). A family history of epilepsy or FS is reported in about 50% of cases (R. Singh et al., 2001). Initial seizures are typically episodes of febrile or afebrile hemiclonic or generalized clonic status epilepticus, although not all patients have status. Other seizure types emerge between one and four years of age, including myoclonic seizures, absence seizures and focal seizures. Interictal EEGs are initially normal, although a photoparoxysmal response may be seen. After the second year, generalized and multifocal spike- and polyspike- wave activity is seen. Seizures are refractory to polytherapy in almost all patients,

although some improvement with topiramate, valproate, benzodiazepines, stiripentol and the ketogenic diet is reported (Chiron et al., 1990; Chiron et al., 2000; Wallace, Wirrell, & Kenney-Jung, 2016). Exacerbation of seizures (particularly myoclonus) and deterioration of neurological function with some sodium channel-blocking drugs such as carbamazepine and lamotrigine are described, although there is debate about whether lamotrigine is truly contraindicated (Brunklaus, Ellis, Reavey, Forbes, & Zuberi, 2012; Dalic, Mullen, Roulet Perez, & Scheffer, 2015; Guerrini et al., 1998).

Most patients continue to have seizures into adult life without a seizure-free period, with seizures changing to a pattern of predominantly brief nocturnal convulsions, often with focal features (Akiyama, Kobayashi, Yoshinaga, & Ohtsuka, 2010; F. E. Jansen et al., 2006). Sensitivity to elevated body and ambient temperature elevation continues into later life (Catarino et al., 2011).

Initial development is normal, but slows in almost all patients in the second year of life, and may regress with episodes of status. Most patients have intellectual disability, this being severe in approximately 50% of children. Neurologic deterioration continues in adulthood (Catarino et al., 2011).

Additional features, including a characteristic progressive deterioration in gait in the second decade of life, are recognized (Gitiaux et al., 2016; Rodda, Scheffer, McMahon, Berkovic, & Graham, 2012). Other late onset features include scoliosis, urinary incontinence and dysphagia (Catarino et al., 2011).

Mortality in Dravet syndrome has been reported as 3.75-15%, one study reporting approximately 15% mortality by 10 years from diagnosis (Cooper et al., 2016; Dravet, 2012; Sakauchi et al., 2011; Skluzacek, Watts, Parsy, Wical, & Camfield, 2011). Causes of death in childhood include sudden unexpected death in epilepsy (SUDEP), and severe brain oedema secondary to prolonged seizures (Cooper et al., 2016). Causes of death in adulthood include SUDEP and respiratory tract infections (Catarino et al., 2011).

When not all features of Dravet syndrome were present (e.g. no generalized spike- wave), it was previously called ‘severe myoclonic epilepsy borderland’ or ‘severe

myoclonic epilepsy borderline’. It has been shown to have a similarly high rate of SCN1A mutations with little meaningful clinical difference, and is now subsumed within the diagnosis of Dravet syndrome (Harkin et al., 2007).

Myoclonic encephalopathy in non-progressive disorders: The syndrome now known as myoclonic encephalopathy in non-progressive disorders has been described by many authors as far back as the mid-1960s (Bureau et al.).

The condition has a female-predominance (2:1), and an average age of onset of 12 months. It is characterized by a long-lasting myoclonic status epilepticus, including both positive and negative myoclonus. Other seizure types, including absences and focal seizure may be seen. The interictal EEG shows a slow and poorly reactive background with prominent paroxysmal abnormalities, typically a monomorphic theta-delta pattern over frontocentral regions and rhythmic delta with admixed spikes seen posteriorly. Seizures are typically refractory to treatment, although some have responded to a combination of valproate and ethosuximide, with an associated improvement in overall condition (Bureau et al.; R. H. Caraballo, Cersosimo, Espeche, Arroyo, & Fejerman, 2007; Lombroso, 1990).

Most have significant developmental impairments and associated neurologic features such as ataxia and movement disorders; dysmorphic features are seen in some.

Early presentations of (typically) childhood-onset epileptic syndromes: A number of epileptic syndromes that most commonly present in early childhood, including MAE (Doose syndrome) and LGS, have an onset age range that includes the late infant period. Although occasional infant-onset cases are seen, these are not common and will not be further discussed here (Stephani, 2006).

‘Structural’ or ‘symptomatic’ focal epilepsies: Approximately one quarter of infants in a recent population-based study had focal seizures associated with a structural brain abnormality (Eltze et al., 2013). These were considered ‘symptomatic focal epilepsy’ (SFE) in the 2001 ILAE classification, and in the most recent classification fit within a variety of groups including ‘distinct constellations’ and ‘epilepsies attributed to and

organized by structural-metabolic causes’, here collectively considered ‘structural focal epilepsy’ (SFE) (Berg et al., 2010; Engel & International League Against, 2001).

Studies have estimated that SFE is seen in 13-28% of patients with epilepsies with onset during the first year of life (D. Battaglia et al., 1999; R. Caraballo, Cersosimo, Galicchio, & Fejerman, 1997; Kramer, 1999) and 12-23% with epilepsies beginning in the first two years of life (Eltze et al., 2013; Okumura et al., 2001). These are likely to be underestimates given the lower quality brain imaging available at the time that most of these studies were performed.

Seizures are typically unifocal, although multiple types of focal seizures may be seen (e.g. in TS) (Pampiglione & Moynahan, 1976). Typically, the epilepsy features depend on the location of seizure onset (+/- spread), and are not consistent across a single aetiology. There are some exceptions to this, listed within the ‘distinct constellation’ group of the 2010 ILAE classification, including gelastic seizures associated with hypothalamic hamartomas and hemifacial spasm associated with cerebellopontine lesions (typically gangliogliomas) (Berg et al., 2010; Berkovic et al., 1988; Gascon & Lombroso, 1971; Harvey & Freeman, 2007; Harvey et al., 1996; Langston & Tharp, 1976).

Many infants evolve to (or from) having epileptic spasms, which can occur during or after infancy (Okumura et al., 1998).

The interictal EEG may be normal. When abnormal, background changes are typically focal background slowing and/or asymmetry with a normal EEG in other regions. IEDs are focal, typically polymorphic and sometimes seen in runs or even continuously (Blume, 1989).

Seizures are commonly refractory to medical therapies, but may cease after epilepsy surgery (Chugani et al., 1988; M. S. Duchowny, Resnick, Alvarez, & Morrison, 1990; Wyllie, Comair, Kotagal, Raja, & Ruggieri, 1996).

Other/complex syndromes: Frequently, epilepsy in infants does not fit into one of the well-defined electroclinical syndromes described above (Deprez et al., 2010; Eltze et

al., 2013; E. Gaily, Lommi, Lapatto, & Lehesjoki, 2016). These infants have ‘complex’ epilepsy phenotypes, often with more than one seizure type.

The term ‘early onset epileptic encephalopathy’ has become widely used to refer to epilepsy with multiple seizure types that has onset in the early months of infancy. However, this term has been used to cover a group of patients with quite disparate phenotypes, including different seizure types and EEG patterns, seizure evolutions and outcomes (Allen et al., 2016; Mastrangelo, 2015; Ohba et al., 2014).

Review of the literature suggests a number of different subgroups amongst this ‘complex group’. Some infants have an EIEE-like epilepsy, with tonic and/or focal seizures beginning in the first few months of life (and often evolution to spasms), without burst-suppression on EEG. A variety of focal/multifocal epilepsies with onset at any age are reported. Others have apparently generalized epilepsies, some being genetic generalise epilepsy-like) (GGE-like) (i.e. with >3Hz generalized spike-wave and seizure types such as myoclonic or absence seizures), and others that are more LGS-like (with multiple seizures types including tonic seizures, generalized and multifocal interictal patterns and generalized ictal rhythms) (Lalani et al., 2014; Ohba et al., 2014; Syrbe et al., 2015).

As more cases are reported, and more descriptions of the epilepsy provided, it is hoped that this group of epilepsies will be able to be further distinguished and classified, which may ultimately be beneficial in predicting cause and prognosis.

Syndrome evolution

Many epileptic syndromes of infancy and childhood have an age-dependent expression, with onset and offset at particular ages that correlated with specific periods of brain development. In infancy and early childhood, age-dependent epileptic syndrome evolution is also seen in some patients. This is best recognized in the transition from EIEE to West syndrome, and then from West syndrome to LGS (Dulac, Nabbout, Plouin, Chiron, & Scheffer, 2007; Yamatogi & Ohtahara, 2002). Similar transitions can be seen in other syndromes, including transition to West syndrome and/or LGS in

patients with structural focal epilepsies, and occasional transient transitions to West syndrome in patients with EME (Djukic et al., 2006; Okumura et al., 1998).

In other infantile epilepsies such as EIMFS and Dravet syndrome, evolution from one epileptic syndrome to another is rarely seen. However, within these syndromes, clear age-related stages of the epilepsy are described (Coppola, 2009; Scheffer, 2012). Evolution of seizure types, and later evolution to established epileptic syndromes, may also be seen in patients with ‘complex’ epilepsy phenotypes, although it remains to be seen whether characteristic patterns of evolution will be further delineated in some of these patients.

Treatment response

A considerable proportion of patients with infantile-onset epilepsies have seizures that persist despite treatment. A prospective community-based cohort of patients with epilepsy onset before age three years found that 50% of patients had intractable seizures, defined as failure of at least two antiepileptic drugs and at least one seizure per month over an 18-month period during the first three years of follow-up (Berg et al., 2004).

Epileptic syndrome is the strongest predictor of treatment response in childhood epilepsy (Berg et al., 2001). In infancy, despite the high rate of overall intractability, there is a wide range of response rates depending on syndrome. Almost all patients with the ‘benign’ familial neonatal and infantile syndromes BFNE, BFNIE and BFIE attain seizure control on monotherapy (Zara et al., 2013). In contrast, almost all patients with EIMFS and EIEE have drug-resistant epilepsy (Coppola, 2009; Yamatogi & Ohtahara, 2002). Other syndromes such as West syndrome have a more variable treatment- response. (Darke et al., 2010).

There is some evidence that non-pharmacologic therapies, namely epilepsy surgery, vagus nerve stimulation and the ketogenic diet, significantly improve seizure control in a subgroup of infants whose seizures are refractory to pharmacologic treatments (R. H. Caraballo et al., 2005; Chugani et al., 1988; Dressler et al., 2015; M. S. Duchowny et

al., 1990; Fernandez, Gedela, Tamber, & Sogawa, 2015; Kossoff, Pyzik, McGrogan, Vining, & Freeman, 2002; Wyllie et al., 1996).

Seizure outcome

The long-term seizure outcome varies with the epileptic syndrome and aetiology. For example, most patients with ‘benign’ familial neonatal and infantile epileptic syndromes become seizure free by the end of infancy, whereas almost all patients with Dravet syndrome have persistent seizures into adult life (Catarino et al., 2011; Grinton et al., 2015; Zara et al., 2013). Persistent seizures of any type in patients with infantile spasms were reported in approximately half of patients by early-mid childhood (Chevrie & Aicardi, 1979; Darke et al., 2010).

A large study of all epilepsies in infants aged 1-12 months reported persistent seizures in 70% at two years and 56% at six years (Chevrie & Aicardi, 1979). No detail was given as to the type of persistent seizures or epileptic syndrome, nor whether they were refractory or well-controlled.

Two similar studies of patients with epilepsy during the first year of life, in which most patients were followed for more than six years, reported seizure cessation rates of 56% (Czochanska et al., 1994; Matsumoto et al., 1983). One study reported that medication withdrawal was achieved in 22% of patients (Czochanska et al., 1994). Rates of seizure freedom were higher in patients with an unknown aetiology for their epilepsy, those with normal intellect, those without additional neurologic impairment, and in boys (Chevrie & Aicardi, 1979). The reason for the difference in seizure outcome between genders in that study is unclear. It did not appear to be due to differences in seizure types, epileptic syndromes or proportion of cases with unknown aetiology, and may be due to statistical variation rather than being a true difference.

1.4.2 Non-epileptic features Comorbid conditions occur frequently in infants with epilepsy, either as other manifestations of the underlying condition or complications of severe neurologic impairment.

Development

Developmental outcomes in patients with infantile epilepsy range from normal to profound intellectual disability. The proportion of infants with developmental impairment, typically defined as a developmental or intellectual quotient of <70, is high. Estimates in cohorts of children with all epilepsies beginning in the first year of life range from 54-78% (Chevrie & Aicardi, 1978; Czochanska et al., 1994; Matsumoto et al., 1983). These figures may be underestimates as some of these groups did not include children with epilepsy onset in the first month or two months of life.

Studies of development in subpopulations of infants with different epileptic syndromes reveal normal development in almost all patients with BFNE/BFNIE/BFIE (Grinton et al., 2015), and almost universal severe developmental impairments in EIEE and EIMFS, highlighting the marked variability in outcome between epileptic syndromes (Coppola et al., 1995; Yamatogi & Ohtahara, 1981). Developmental outcomes vary from normal to severely impaired in infants with infantile spasms, although there remains a very high chance of abnormal development, with some degree of impairment in 70-80% of patients (Chevrie & Aicardi, 1971; Darke et al., 2010; Koo et al., 1993). An ‘idiopathic’ subgroup of infants with IS who have normal developmental outcomes has long been recognized, these infants having normal development at epilepsy onset and prompt resolution of spasms (Kivity et al., 2004). Thus, there are some epileptic syndromes with a high likelihood of normal developmental outcome, and some with a high likelihood of an abnormal outcome, albeit with significant variability in severity of impairment.

The strongest predictor of normal versus abnormal developmental outcome is said to be the underlying aetiology of the epilepsy rather than the characteristics of the epilepsy itself (Rantala & Ingalsuo, 1999). While this is true for some infants with aetiologies in which development delay is expected, even if epilepsy is promptly diagnosed and successfully treated, such as trisomy 21, it is becoming increasingly clear that it does not hold for other aetiologies (Eisermann et al., 2003). An example of this is seen with some sodium and potassium channelopathies, where both normal and markedly abnormal developmental outcomes can be seen with mutations in the same gene such as SCN1A, SCN2A and KCNQ2 (Heron et al., 2002; Howell et al., 2015; N. A. Singh et al.,

1998; Weckhuysen et al., 2012). Finally, many infants have an unknown aetiology; aetiology can obviously not be used as a predictor of outcome here (Eltze et al., 2013; E. Gaily et al., 2016).

In addition to aetiology, the epilepsy itself – that is, the nature of the seizures and EEG abnormalities - contribute to neurodevelopmental outcome. This is the concept of an ‘epileptic encephalopathy’, defined by the ILAE as being “the notion that the epileptic activity itself may contribute to severe cognitive and behavioural impairments above and beyond what might be expected from the underlying pathology alone and that these might worsen over time” (Berg et al., 2010).

There are many epilepsy-related factors associated with poorer outcome. The type of seizures may predict the degree of developmental impairment relative to that expected from the underlying aetiology. For example, infants with TS and IS have a poorer outcome than those with TS without IS. The age at epilepsy onset also appears important, with early seizure onset associated with poorer outcome (H. Freitag & Tuxhorn, 2005; O'Callaghan et al., 2011; Vasconcellos et al., 2001; Vendrame et al., 2009; Zaroff, Devinsky, Miles, & Barr, 2004). Further, pharmacoresistant epilepsy in the first year of life predicts poor outcome, with an 87% chance of IQ<80 (Berg, Zelko, Levy, & Testa, 2012). VABS scores decline over time in those with pharmacoresistant epilepsy with onset before age three years, but not in those with treatment-responsive epilepsy (Berg et al., 2004).

While the aforementioned factors could be considered markers of a more severe condition rather than the result of seizures and EEG abnormalities on development, data from infants with delayed diagnosis and/or delay to treatment highlights the link between the duration of exposure to seizures and EEG abnormalities with poor developmental outcome. Patients with Down syndrome and IS treated within two months of seizure onset had a median IQ of 37, compared with 14 in those treated later than two months (Eisermann et al., 2003). In patients with IS of any aetiology, multiple studies have shown poorer developmental outcome in those with prolonged lead-time to treatment (Kivity et al., 2004; Matsumoto et al., 1981; O'Callaghan et al., 2011). Berg et al looked more broadly at a community-based cohort of children with early onset

epilepsies (onset under three years old), showing a lower score on the VABS in those with a delayed presentation compared with those with prompt presentation (and prompt commencement of treatment). This difference was evident at diagnosis and persisted over many years, suggesting that even a relatively short period of seizures (delayed presentation was considered to be >1 month) can have long-term impacts (Berg, Loddenkemper, & Baca, 2014). Finally, a longer duration of epilepsy before epilepsy surgery was negatively correlated with developmental quotient in infants with uncontrolled focal epilepsy who underwent hemispherectomy (Cormack et al., 2007; D'Argenzio et al., 2011; Jonas et al., 2004; Loddenkemper et al., 2007). Seizure cessation with epilepsy surgery before age three years results in an improvement in mental age in all patients. Most important to note, is that early surgery in infants (<1 year old) resulted in a median 30-point increase in DQ (compared with a median 0 point change in those with surgery between 1-3 years old), suggesting that some brain function is recoverable if the epileptic activity is terminated early. Even more compelling is data from the TS literature showing that instituting AED prior to epilepsy onset if the EEG becomes epileptiform (compared with institution of treatment within a week of epilepsy onset) reduces the rates of drug resistant epilepsy and markedly improves developmental outcome, suggesting that prevention of seizures confers even more benefit (Jozwiak et al., 2011).

Finally, epilepsy treatment can also adversely impact development. Both animal and human data reveal impacts of AEDs on the developing brain. Phenobarbitone use in infancy for febrile seizure prophylaxis is associated with a permanent lowering of IQ by 5-10 points (Farwell et al., 1990). Children of mothers taking sodium valproate in pregnancy have lowered IQ (Nadebaum et al., 2011). There is no data looking at the magnitude of effect of anticonvulsant polytherapy on developmental outcome in infancy, as such an effect is difficult to disentangle from that of seizures, EEG abnormalities and the underlying condition.

With current therapies, maximizing developmental outcomes in epilepsy relies on effecting seizure freedom and EEG normalization at the earliest possible time to minimize the detrimental effects of the epilepsy. This requires rapid referral and diagnosis, and prompt institution of appropriate treatment. Delays to diagnosis are

common, one study reporting delays of more than one month in 41% of children with early onset epilepsies. A variety of reasons for delay were reported, including parents not recognizing episodes as potential seizures, problems with scheduling appointments and missed or deferred diagnosis by medical professionals (Berg et al., 2014). Another important factor is the delayed utilization of epilepsy surgery in infants with a surgically-remediable refractory epilepsy (Harvey, Cross, Shinnar, Mathern, & Taskforce, 2008; Pestana Knight et al., 2015). Improving rates of timely diagnosis and appropriate use of currently available therapies are a priority.

Although these approaches will make a significant difference in some patients, seizure freedom is not attained with current therapies in many infants, even when they receive early, appropriate therapy. As such, there is an urgent need for novel therapeutic interventions. Further, most current therapies do not target the underlying aetiology. Some aetiologies may not be amenable to such targeted therapies, yet where disease- modifying (rather than symptomatic) treatments could be developed in the future, these would be expected to have a larger impact on developmental outcome, and should be a focus of future research.

In addition to developmental and cognitive impairments, autism spectrum disorders (ASD) were seen in 17.6-35.5% of patients with a history of infantile spasms, 10% of infants with epilepsy onset under two years old and 7.1% of patients with seizures other than spasms in the first year of life (Berg, Plioplys, & Tuchman, 2011; Saemundsen, Ludvigsson, Hilmarsdottir, & Rafnsson, 2007; Saemundsen, Ludvigsson, & Rafnsson, 2007). Interestingly, the major factor associated with autism in children with epilepsy is intellectual disability; the risk of ASD is substantially higher in those with intellectual disability than those with normal IQ (Amiet et al., 2008; Berg et al., 2011). The mechanisms that explain the co-occurrence of epilepsy, intellectual disability and ASD are not clear, but likely include similar factors to those that account for intellectual disability alone, namely the underlying aetiology and damaging effects of the epilepsy. Evidence for the former comes from the large number of aetiologies in which either or both epilepsy and autism are typical features, presumably highlighting that particular pathophysiologic mechanisms can produce both conditions. Further study is needed to definitively demonstrate the latter. Just a single study has investigated whether early

effective treatment of seizures results in a reduced rate of ASD. In a group with infantile spasms, the authors reported that early diagnosis and successful treatment did not reduce the rate of autism (Bitton et al., 2015). However, a number of the children in the successfully treated group had ongoing epileptiform EEG despite seizure cessation. Thus, the question of whether early complete resolution of all epileptic features (i.e. seizures AND EEG abnormality) reduces ASD is still open.

Finally, rates of other neurobehavioural and neuropsychiatric disorders are substantially higher than population rates. Significant attention deficits are seen in over 40% of patients with severe epilepsies that had onset before age three (Sherman, Slick, Connolly, & Eyrl, 2007). One study reported psychiatric disorders in 27.6% of survivors of infantile spasms (Riikonen, 1982).

Neurologic

A broad range of neurologic impairments is seen in conjunction with, or following, infantile epilepsy.

Czochanska et al reported microcephaly in 8% of survivors of epilepsy in the first year of life, presumably being a feature of the underlying conditions (Czochanska et al., 1994). Neurologic abnormalities including hemiplegia, bilateral pyramidal signs and hypotonia were present in 31% of a cohort of infants with epilepsy that began in the first year of life (Chevrie & Aicardi, 1978). Other neurologic abnormalities, including movement disorders, ataxia, autonomic dysfunction and hearing impairment are reported in some infantile epilepsies, although the rate at which these occur across the whole spectrum of infantile epilepsies is unknown (Campeau et al., 2014; Kobayashi et al., 2016; Panagiotakaki et al., 2010; Syrbe et al., 2015). Their manifestations as part of specific genetic aetiologies are increasingly recognized, and are listed in later in the chapter and in Appendix A.

Non-neurologic

Non-neurologic comorbidities are also seen frequently, although the rates of each have not been determined in this patient group. These include co-existent dysmorphism,

cutaneous abnormalities and malformations of other organs, and sequelae of seizures and neurologic impairment including failure to thrive, growth failure, requirement for supplemental feeding, gastro-oesophageal reflux disease and aspiration pneumonia.

1.4.3 Survival Three large hospital-based cohorts of patients with epilepsy in the first year of life followed for at least one, six and three years reported survival rates of 89%, 85% and 89% respectively (Chevrie & Aicardi, 1978; Czochanska et al., 1994; Matsumoto et al., 1983). It is possible that these figures may be an underestimate of death in the early years of life in patients with infantile epilepsy (or at least those severe enough to be managed in a hospital setting), as two studies did not include the youngest patients. A population-based study which did include neonates showed a higher rate of death in epilepsies with neonatal onset compared with those with onset in later infancy (Moseley, Wirrell, Wong-Kisiel, & Nickels, 2013). A lower rate of death (4.4%) was reported in a second population-based study of epilepsy in the first year of life, with infants followed until two years old (E. Gaily et al., 2016). This lower figure compared with that in the hospital-based cohorts may reflect a greater proportion of infants with ‘benign’ epilepsies who may not have been included in those cohorts, although the reasons for the discrepancy between this study and the other population-based study are not clear.

Most deaths in infantile and severe early childhood epilepsy are due to respiratory infection and other complications of neurologic impairment, with a smaller proportion related directly to the epilepsy, such as SUDEP and refractory status epilepticus (Berg et al., 2013; Chevrie & Aicardi, 1978; Czochanska et al., 1994; Harvey, Nolan, & Carlin, 1993; Matsumoto et al., 1983; Morse & Kothare, 2016; Moseley et al., 2013). Some infants with particular aetiologies, particularly SCN1A mutations are at higher risk of SUDEP; this may also be the case in other sodium channelopathies (Cooper et al., 2016; Howell et al., 2015; Larsen et al., 2015; Sakauchi et al., 2011; Veeramah et al., 2012). In SCN1A, the function of sodium channels has been demonstrated to be abnormal in both brain and heart tissue. The relative contribution of the channels in each organ to SUDEP is not clear (Auerbach et al., 2013; Kalume et al., 2013).

1.5 Aetiologies of the infantile epilepsies

1.5.1 Classification of aetiologies Many aetiologies have been identified for infantile-onset epilepsies. In considering diagnoses in the individual patient, as well as mechanisms by which epilepsy arises, there is a need to classify this large number of aetiologies.

A number of classifications exist and, over time, the most widely used classification has changed in response to improved understanding of the neurobiology. Earlier classifications attempted to divide aetiologies into ‘prenatal vs perinatal vs postnatal’ groups, depending on the timing of the ‘insult’, which at that time, what presumed acquired in most cases. With the increasing recognition of the existence of both genetic and acquired aetiologies, as well as evolution in nomenclature for unknown aetiologies, classifications have changed - initially to the ‘symptomatic vs idiopathic vs cryptogenic’ scheme used by the ILAE from 1989 to 2006 and, more recently, to the current ILAE classification groups of structural, metabolic, genetic and unknown (Berg et al., 2010; Chevrie & Aicardi, 1977; ILAE, 1989). Here, the genetic group is primarily used to refer to genetic disorders that do not have a malformative or metabolic basis. Malformative disorders with a genetic basis such as TS are usually categorized as ‘structural’, with recognition of overlap with the genetic group. In this study, when this classification is used, the group will be referred to as ‘genetic’ (in inverted commas) to avoid confusion with use of the same term to refer to any condition with a genetic basis.

Changes to the classification reflect more efficient or natural categorisations that occur with advances in understanding. It is likely that further changes will occur, and that these may include:

 Major subgrouping change to ‘genetic vs acquired’.

o It is already recognized that many structural and almost all metabolic conditions have a genetic basis, so there is overlap accepted in the current classification (Berg et al., 2010) o It is important to note that ‘acquired’ conditions are not necessarily ‘non- genetic’, but they do require non-genetic factors for expression of the

condition. Some ‘acquired’ conditions do have a genetic component that predisposes to either the initial condition or the epileptogenesis that follows the initial condition, further discussed in section 2.5.2 (Shah et al., 2012; S. Y. Zhang & Casanova, 2015)

 A move to a mechanism-based subgrouping within the genetic group (or possibly across all groups).

o Subgroupings may include channelopathies, disruption of neurotransmission, disruption of a particular molecular pathway etc. (rev in (McTague et al., 2013)).

1.5.2 Acquired aetiologies Infantile epilepsy is described following a variety of acquired insults to the cerebral cortex in the pre-, peri- and early postnatal periods. Insults in early fetal development often result in a predominantly malformative lesion, which can be focal as seen in schizencephaly after vascular injury (Shah et al., 2012; Yoneda et al., 2013), or more diffuse as seen with polymicrogyria (PMG) following congenital cytomegalovirus infection (Lehmann, 1982). Insults in later fetal development or the postnatal period typically result in predominantly destructive lesions, but may interfere with postnatal developmental processes. Examples of these insults include focal insults such as arterial ischaemic stroke (Wanigasinghe et al., 2010), and multifocal or diffuse insults such as HIE (HIE) (Inoue et al., 2014), neonatal hypoglycaemia (Udani, Munot, Ursekar, & Gupta, 2009) and infections or inflammatory processes of the central nervous system (Riikonen, 1993).

A recent population-based study identified acquired insults as the cause in 16% of patients with epilepsy beginning under two years old, with the major causes being central nervous system infections, and hypoxic-ischaemic encephalopathy (Eltze et al., 2013). Previous studies identified a higher proportion of ‘perinatal’ aetiologies for epilepsy in the first year of life than the more recent study, which is probably partly attributable to improvements in obstetric care, and partly that a perinatal cause is now less likely to be assumed where the aetiology is unknown (Chevrie & Aicardi, 1977; Czochanska et al., 1994; Matsumoto et al., 1983). The proportion of infants with

acquired aetiologies differs across populations, being higher in developing countries with higher rates of conditions such as neonatal hypoglycaemia and hypoxic-ischaemic encephalopathy (Chawla, Aneja, Kashyap, & Mallika, 2002; Kwong, Chak, Wong, & So, 2001; Udani et al., 2009).

Most cortical insults in the developing brain are potentially epileptogenic. The relative contribution of the timing, location, and specific type of pathology to the likelihood of developing epilepsy, timing of seizure onset and type of epilepsy presentation, is unclear. The infantile period is a high-risk time for epilepsy onset in patients with early acquired brain lesions. Where large groups of infants with a single type of acquired insult have been studied, infantile epilepsy occurred in 22% of patients with perinatal arterial ischaemic stroke within the first year of life, and 34% of patients with neonatal hypoglycaemia before age two years (Burns, Rutherford, Boardman, & Cowan, 2008; Wanigasinghe et al., 2010). Like in older people, the epilepsy following an acquired early life insult typically presents following a silent period. Where the timing of the insult was clearly during the neonatal or early infantile period, the epilepsy presentation occurred after a mean of 5.1 months (Guggenheim, Frost, & Hrachovy, 2008). Infantile seizure presentations of acquired aetiologies are most commonly with spasms (Watanabe, Hara, Miyazaki, & Hakamada, 1980).

Genetic factors likely play a role in some acquired insults, either with respect to the risk of acquiring the insult or to the risk of epileptogenesis. With respect to the former, a number of genes including SUR1 and Kir6.2 are associated with hyperinsulinaemia and neonatal hypoglycaemic brain injury. Similarly, genes in the TLR3 pathway are associated with risk of herpes simplex virus encephalitis (Kapoor, James, & Hussain, 2009; Kumaran, Kar, Kapoor, & Hussain, 2010; S. Y. Zhang & Casanova, 2015). Some studies report higher rates of epilepsy after brain insults in those with a family history of epilepsy, suggesting a possible contribution of genetic factors to post-insult epileptogenesis (Burns et al., 2008).

1.5.3 Genetic and presumed genetic aetiologies As discussed above, the current classification of aetiologies contains genetic or presumed genetic causes of epilepsies across all groups (structural-metabolic, genetic

and unknown). While this classification is artificial when considering mechanisms of disease, it remains useful for considering an approach to diagnostic evaluation given currently available diagnostic techniques, and is likely to remain useful in this respect (particularly for structural aetiologies) even when genetic testing is more widespread. In this section, the structural (non-acquired), metabolic and genetic aetiologies of infantile epilepsy are outlined in the tables below.

Brain malformations, including malformations of cortical development and hindbrain malformations are well-described structural causes of infant epilepsy. Many of these have a known or presumed genetic basis (Barkovich, Millen, & Dobyns, 2009; Parrini, Conti, Dobyns, & Guerrini, 2016). Discovery of the causative genes has highlighted common pathologic mechanisms; mutations in genes in the mammalian target of Rapamycin (mTOR) pathway can cause cortical dysplasias and TS, mutations in genes involved in microtubule function can cause lissencephaly and microcephaly (Crino, 2015; Parrini et al., 2016). Mutations that occur post-zygotically (i.e. ‘somatic’ mosaic) may be present in brain tissue; these are increasingly recognized as important in brain malformations (further discussed below) (Jamuar et al., 2014; Lee et al., 2012; Poduri, Evrony, et al., 2012; Shirley et al., 2013). Brain malformations that can cause infant epilepsy and their genetic bases are listed in Table 1.1. Although some are not strictly malformative, white matter abnormalities are included in this table given they can be detected (i.e. macroscopically) on brain imaging (Schiffmann & van der Knaap, 2009).

Table 1.1 Structural (non-acquired) causes of infant epilepsy

Type of structural abnormality Pathway(s) and genes

Prosencephalic development

Holoprosencephaly SHH, SIX3

Cortical development

Abnormal cell proliferation or apoptosis

Microcephalies Tubulins: TUBA1A, TUBB2B, TUBB3, TUBG1 Microtubule-associated proteins: DYNC1H1, KIF5C, NDE1 Cellular trafficking and signalling: RAB18, RAB3GAP1, RAB3GAP2, STAMBP, TBC1D20 Other: PNKP, RNU4ATAC, WDR62

Abnormal proliferation (including tuberous PI3K-AKT-MTOR pathway: AKT1, AKT3, DEPDC5, sclerosis and other focal cortical dysplasia MTOR, NPRL2, NPRL3, MTOR, PIK3CA, PIK3R2, type II spectrum disorders) TSC1, TSC2 Other: CCND2, CNTNAP2, FGFR1, BRAF

Megalencephaly +/- dysgenesis (including PI3K-AKT-MTOR pathway: AKT3, MTOR, PI3KCA, focal cortical dysplasia and polymicrogyria) PIK3R2, PTEN, STRADA RAS-MAPK pathway: BRAF, HRAS, KRAS, MAP2K1, MAP2K2, NRAS

Abnormal migration

Lissencephaly-pachygyria-band heterotopia Tubulins: TUBA1A, TUBA8, TUBB2B, TUBG1 spectrum Microtubule-associated-proteins: PAFAH1B1 (LIS1), DCX, DYNC1H1, KIF2A, KIF5C, NDE1 Other: ARX, NSDHL, RELN, VLDR

Cobblestone malformations Alpha-dystroglycanopathies: FKRP, FKTN, ISPD, LARGE1, POMGNT1, POMT1, POMT2 Other: GPR56

Periventricular nodular heterotopias ARFGEF2, FLNA, NEDD4L

Post-migrational development

Polymicrogyria and schizencephaly COL18A1, EMX2, FIG4, KIAA1279, OCLN

Other

Porencephaly (+/- other abnormalities) Collagens: COL4A1, COL4A2

Sturge-Weber syndrome GNAQ

Incontinentia pigmenti IKBKG

Subcortical development

Hypothalamic hamartoma Sonic hedgehog pathway: GLI3

Midbrain/hindbrain malformations

Type of structural abnormality Pathway(s) and genes

Cerebellar +/- other hindbrain enlargement HRAS

Cerebellar hypoplasia/agenesis OPHN1, PTF1A

Pontocerebellar hypoplasia RNA splicing endonuclease subunit genes: TSEN2, TSEN34, TSEN54 Other: AMPD2, CASK, EXOSC3, PCLO, RARS

Joubert syndrome CC2D2A, OFD1

Brainstem malformations OTX2

Abnormalities of white matter Absent corpus callosum (+/- other C12ORF5, DHCR24, EPG5, HCCS, MED12, OTX2 abnormalities)

Hypomyelination HSPD1, TUBB4A

Dysmyelination EIF2B1, EIF2B2, EIF2B3, EIF2B4, EIF2B5, GFAP, HEPACAM, MLC1, RNASET2

See Appendix D and text in this chapter for gene references

Many inborn errors of metabolism, involving a variety of small and large molecules and biochemical pathways, are reported to cause infantile epilepsy. Epilepsy is a common or universal feature of some metabolic disorders, such as non-ketotic hyperglycinaemia and the pyridoxine-related epilepsies, and a rare association in others (rev in (Wolf, Garcia-Cazorla, & Hoffmann, 2009)). Metabolic causes of infant epilepsy are listed in Table 1.2.

Table 1.2 Metabolic disorders associated with infantile epilepsy

Type of metabolic abnormality Genes

Small molecule disorders

Amino and organic acid disorders Canavan disease: ASPA Congenital glutamine deficiency: GLUL DBT Ethylmalonic aciduria: ETHE1 Fumarase deficiency: FH Glutaric aciduria: D2HGDH Maple syrup urine disease: BCKDHA, BCKDHB, DBT Molybdenum cofactor deficiency: GPHN, MOCS1, MOCS2 Phenylketonuria: PAH Propionic acidaemia: PCCA, PCCB Sulphite oxidase deficiency: SUOX

Neurotransmitter disorders Disorders of GABA metabolism: ABAT, ALDH5A1 Glycine encephalopathy: AMT, GCSH, GLDC Serine synthesis defects: PHGDH, PSAT1, PSPH Tetrahydrobiopterin disorders: GCH1, PTS, QDPR, SPR

Disorders of vitamin metabolism Pyridoxine-related epilepsies: ALDH7A1, ALPL, PNPO Biotin-related epilepsies: BTD, HLCS Vitamin B12 metabolism: HCFC1, MMACHC, MMADHC, MTR Folate metabolism: FOLR1, MTHFR

Disorders of mitochondrial function Coenzyme Q10 deficiencies: COQ2, COQ4, COQ6, COQ9, PDSS2 Mitochondrial depletion disorders: FBXL4, POLG, RRM2B, SUCLA1, SUCLG1 Oxidative phosphorylation defects: AARS, ATP5A1, BCS1L, BOLA3, COX10, COX15, EARS2, FARS2, FASTKD2, FOXRED1, GFM1, GTPBP3, LIAS, NARS2, NDUFA1, NDUFA10, NDUFA11, NDUFA2, NDUFAF2, NDUFAF3, NDUFAF4, NDUFAF5, NSUFS1, NDUFS2, NDUFS4, NDUFS6, NDUFS7, NDUFS8, NDUFV1, NUBPL, PET100, RMND1, SCO1, SCO2, SDHA, SURF1, TMEM70, TSFM Pyruvate dehydrogenase deficiency: PDHA1, PDHX

Disorders of glucose metabolism and Glut-1 deficiency: SLC2A1 transport Diabetes-associated genes: IER3IP1, KCNJ11 Hypoglycaemia-associated genes: GLUD1

Other Creatine disorders: GAMT, SLC6A8 Purine metabolism: ADSL, HPRT1 Metabolism of metals: ATP7A

Large molecule disorders

Congenital disorders of glycosylation CDG type I: ALG1, ALG11, ALG12, ALG13, ALG2, ALG3, ALG6, ALG8, ALG9, DOLK, DPAGT1, DPM1, DPM2, MPDU1, PMM2, RFT1

Type of metabolic abnormality Genes

CDG type II: COG4, COG6, COG7, COG8, MOGS, SLC35A2

Lysosomal storage disorders Gangliosidoses: GLB1, GM2A, HEXA, HEXB Sphingolipidoses: GALC, GBA, PSAP Neuronal ceroid lipofuscinoses: CLN3, CTSD, PPT1, TPP1

Disorders of glycoprotein metabolism Fucosidosis: FUCA1 Mannosidosis: MANBA Schindler disease: NAGA

Perioxisomal disorders Biogenesis defects: PEX1, PEX7 Peroxismal enzyme deficiencies: ACO1, HSD17B4

Other Congenital disorder of deglycosylation: NGLY1 Lafora disease:EPM2A

See Appendix D and text in this chapter for gene references

‘Genetic’ causes of infant epilepsy can be divided into chromosomal conditions (Table 1.3) and single gene disorders (Table 1.4). For some chromosomal conditions, the gene critical to development of epilepsy has been identified; in some cases, such as for the CHD2 gene within the 15q26.1 deletion, identification of the critical gene has led to the discovery of intragenic mutations in that gene (Carvill, Heavin, et al., 2013).

Table 1.3 Chromosomal abnormalities associated with infant epilepsy

Chromosomal Critical gene Ref abnormality Monosomy 1p36 Unknown Shapira 1997 (Postulated: GABRD, KCNAB2, CHD5) Bahi-Buisson 2008 Shimada 2015 1q43q44 deletion Unknown Ballif 2012 (Critical region involves FAM36A, Speevak 2013 HNRNPU, HNRNPU-AS1) 2q23.1 deletion MBD5 Rev in Motobayashi 2012 2q24 deletion/duplication Sodium channel gene cluster Rev in Davidsson 2008 (SCN1A/2A/3A) 4p16.3 deletion (Wolf- Unknown Wolf 1965 Hirschhorn syndrome) (Critical region involves ZNF721, PIGG, Hirschhorn 1965 ABCA11P) Battaglia 2009 Ho 2016 5q14.3 deletion MEF2C Le Meur 2010 Paciorkowski 2013 5q31.3 deletion PURA Shimojima 2011 Brown 2013 Lalani 2014 Hunt 2014 7q11.23q21.11 deletion MAGI2^ Mizugishi 1998 (Williams syndrome +) Marshall 2008 Roethlisberger 2010 9q33q34 deletion STXBP1, SPTAN1 Saitsu 2008 Tetrasomy 12p (Pallister- Unknown Pallister 1977 Killian syndrome) Killian 1981 Giordano 2012 14q12 deletion/duplication FOXG1 Ariani 2008 Yeung 2009 Seltzer 2014 Ring 14 Unknown Gligenkrantz 1971 (Postulated: FOXG1) Giovannini 2013 15q duplication syndrome UBE3A, GABRB3 Battaglia 1997 (includes isodicentric(15)) Battaglia 2008 Conant 2014 Finucane 2016 15q11q13 deletions - UBE3A, GABRB3 Knoll 1989 maternal copy (Angelman Viani 1995 syndrome) Laan 1997 Minassian 1998 15q13.3 deletion (BP4-5) CHRNA7 Sharp 2008 Helbig 2009 Masurel-Paulet 2010 15q26.1 deletion CHD2 Veredice 2009 Dhamia 2011 Capelli 2012 Carvill 2013

Chromosomal Critical gene Ref abnormality Thomas 2015 16p11.2 duplication/deletion Unknown Shinawi 2010 (?PRRT2) Dale 2011 Mefford 2011 Reinthaler 2014 Steinman AJMG-A 2016 16p13.11 deletion/duplication NDE1* De Kovel 2010 Heinzen 2010 17p11.2 deletion (Smith- Unknown Roccella 1999 Magenis syndrome) Vlangos 2005 Goldman 2006 Hino-Fukuyo 2009

17p13.3 deletion (Miller- PAFAH1B1 Dobyns 1983 Dieker syndrome) Gastaut 1987 Herbst 2016 17q21.31 deletion (Koolen- KANSL1 Koolen 2006 De Vries syndrome) Koolen 2008 Koolen 2012 Zollino 2015 Trisomy 18 (Edwards Unknown Kumada 2013 syndrome) 18q deletion Unknown Rev in Verrotti 2015 Ring 20 Unknown Augustijn 2001 (Postulated: KCNQ2) Walleigh 2013 Vignoli 2016 20q13 deletion KCNQ2 Kurahashi 2009 Trisomy 21 (Down Unknown Rev in Arya 2011 syndrome) (Postulated: KCNJ6) Blichowski 2015 Joshi 2016 22q11.2 deletion Unknown Kim 2016 Xq28 duplication MECP2 Van Esch 2005 Vignoli 2012 Caumes 2014 Lim 2016

^Conflicting evidence of role of MAGI2 in epilepsy associated with 7q11.23q21.11 deletion *NDE1 considered critical gene for the neurodevelopmental phenotype in the 16p13.11 deletion, but its role in epilepsy specifically is not clear

The number of single gene disorders causing infantile epilepsy is now large. A number of cellular pathways and functions is implicated in the genetic epilepsies as noted earlier in this section. Disorders of ion channel function and synaptic function are well- recognised, with multiple genes involved in these functions reported (rev in (McTague, Howell, Cross, Kurian, & Scheffer, 2016)).

Some genes have been reported only in small numbers of patients, others such as SCN1A, CDKL5 and STXBP1, are very well described, with over 100 cases of each reported (Bahi-Buisson, Kaminska, et al., 2008; Bahi-Buisson, Nectoux, et al., 2008; Brunklaus et al., 2012; Claes et al., 2001; Kalscheuer et al., 2003; Saitsu et al., 2008; Stamberger et al., 2016). The phenotypic features of these more widely-reported genes are well-described, and some can be recognized by experienced clinicians.

More detailed phenotypic information on the single gene disorders causing ‘severe’ infantile epilepsies is provided in Appendix A (adapted from (McTague et al., 2016)).

Table 1.4 Single gene (non-malformative, non-metabolic) disorders causing infantile epilepsy

Cellular function or pathway disrupted* Genes

CACNA1A, FHF1, KCNA2, KCNB1, KCNQ2, Channelopathies – voltage-gated channels and KCNQ3, KCNT1, KCTD7, SCN1A, SCN2A, regulators of SCN8A GABRA1, GABRB3, GABRG2, GRIN1, GRIN2A, Channelopathies – ligand-gated channels GRIN2B, HCN1 Synaptic function – pre-synaptic compartment DNM1, SLC6A1, STXBP1, TBC1D24 ARHGEF9, DOCK7, GNAO1, IQSEC2, PLCB1, Synaptic function – post-synaptic compartment SYNGAP1 Transporters SLC1A2, SLC12A5, SLC13A5, SLC25A22, ARX, BRAT1, CDKL5, CHD2, FOXG1, MEF2C, DNA transcriptional regulation, replication and repair PNKP, PURA, SIK1, SMC1A AARS, ALG13, EEF1A2, PIGA, PIGO, QARS, Protein translation and modification SLC35A2, UBA5 KIAA2022, PCDH19,SPATA5, SPTAN1, Other WDR45, WWOX Unknown function PRRT2, ROGDI, SETBP1, SZT2 See Appendices A and D, and text in this chapter, for gene references

1.5.4 Advances in knowledge following identification of genetic causes of infantile epilepsies

Identification of the genetic basis of some of the infantile epilepsies in which the cause was previously unknown has led to advances in understanding of the mechanisms and spectra of this group of conditions (rev in (McTague et al., 2016)), including:

 recognition that some genetic epilepsies are not ‘inherited’, with major roles identified for de novo dominant and somatic mutations, and subsequent implications for approaches to gene discovery, development of novel genetic diagnostic techniques and reproductive counselling  expansion in both genotypic and phenotypic spectra, revealing a complex landscape of disease-gene associations  an understanding of age- or developmental-stage-specific expression of some genes, and the recognition that this may correspond to age-related presentations of disease

Further, this knowledge forms the beginnings of an understanding of the molecular pathways important in epileptogenesis in the developing brain, and will be important in guiding the development of novel and targeted therapeutic approaches (Consortium et al., 2013; Paciorkowski, Thio, & Dobyns, 2011).

Mutation type and inheritance patterns

A broad range of mutation types and inheritance patterns is described in genetic infantile epilepsies.

The majority of reported mutations to date are, like in most diseases, exonic, although some splice site and other intronic mutations are described (Depienne, Trouillard, et al., 2009; McTague et al., 2016; Weber, Kreth, & Muller, 2016). A variety of missense, frameshift and nonsense mutations are reported, with variable effects on transcription and translation, typically resulting in production of an altered amount or altered type of protein product. Mutations can have differential effects, with loss of function, dominant negative and gain of function mutations reported (Brunklaus et al., 2012; S. Jansen et al., 2016; Milligan et al., 2014; Orhan et al., 2014).

Autosomal dominant, autosomal recessive, X-linked and mitochondrial inheritance patterns are all reported in infantile epilepsies (Kalscheuer et al., 2003; Poduri, Chopra, et al., 2012; Rahman, 2012). Most of the ‘benign’ infant epilepsies have an autosomal dominant inheritance pattern with multiple affected family members (Zara et al., 2013). In the ‘severe’ infantile epilepsies, most affected individuals will not reproduce. Therefore, autosomal dominant inheritance usually only occurs in the context of epilepsies of variable severity or penetrance (e.g. SCN1A with a family with genetic epilepsy with febrile seizure plus (GEFS+) and a child with Dravet syndrome) (Nabbout et al., 2003)or when inheritance occurs from an unaffected (or mildly affected) parent with somatic or germline mosaicism (Milh et al., 2015; Xu et al., 2015; Zerem et al., 2014). Parental mosaicism is an increasingly recognized phenomenon, reported in up to 10% of parents, this having major implications for reproductive counselling.

Increasingly, de novo dominant mutations (a new mutation in the child not present in either parent) are recognized as a major cause of disease in the infantile epilepsies (rev

in (McTague et al., 2016)). These de novo mutations are probably present in the gamete or occur in very early embryogenesis in many cases given they are present in tissues of multiple lineages (Vadlamudi et al., 2010). It is now also recognized that somatic mosaicism can occur relatively later in embryogenesis, resulting in mutations confined to particular regions of the body or tissue lineages, and phenotypes such as focal brain malformations (Poduri, Evrony, et al., 2012). Somatic mosaicism is likely an under- recognised mechanism of neurologic disease. However, detection of somatic mosaicism causing phenotypes referable to the central nervous system is problematic as identifying a mutation requires sampling brain tissue.

Genotype-phenotype variability

A recurring theme in genetic discovery in epilepsy is the concept of an expanding phenotypic spectrum – that is, a particular phenotype is initially described to be associated with a gene and, over time, mutations in the same gene are also found in infants with different phenotypes – including both those with similarities in the epilepsy, but significant differences in severity (e.g. SCN1A, SCN2A, KCNQ2), and those with very different epilepsy or other phenotypes (e.g. TBC1D24). As such, for many genes, the phenotypic spectrum is relatively broad (Campeau et al., 2014; Claes et al., 2001; Corbett et al., 2010; Escayg et al., 2000; Falace et al., 2010; Heron et al., 2002; Howell et al., 2015).

The reasons for this variability and complexity is often unclear (and likely differs from gene to gene), but may include factors related to the type of mutation (including variation in the amount of protein produced, differential impact on critical sites or binding partners of the protein product), timing of (de novo) mutations, timing and location of gene expression, epigenetic factors and modifier genes (rev in (McTague et al., 2016).

Some examples of phenotype-genotype variability for which the molecular basis has been examined include:

 ARX. Truncation and missense mutations in the DNA binding domain produce malformative phenotypes, and non-malformative phenotypes are caused by

polyalanine repeat expansions or missense mutations outside the DNA binding domain (Shoubridge, Fullston, & Gecz, 2010)  KCNT1. Disease-causing mutations are associated with gain of function. The degree of gain of function correlates with disease severity (EIEE>EIMFS>severe ADNFLE) (Milligan et al., 2014)  SCN8A. Gain-of-function mutations cause severe epilepsies, loss-of-function mutations cause intellectual disability without epilepsy (O'Brien & Meisler, 2013)

The reverse phenomenon is also true, that is, that one epileptic syndrome is associated with mutations in multiple genes. This is true to varying degrees for all infantile epileptic syndromes, with high variability in the genetic aetiologies of West syndrome and significantly lower variability in Dravet syndrome, in which SCN1A mutations predominate (Depienne, Trouillard, et al., 2009; Djemie et al., 2016; Michaud et al., 2014). As knowledge of phenotypes of particular genes increases, phenotypic differences are beginning to be identified among different genes that cause the same epileptic syndrome, including epilepsy factors such as evolution of the epilepsy over time and response to particular treatments, and presence or absence of particular comorbidities (Howell et al., 2015; Kim et al., 2013; Stamberger et al., 2016; Weckhuysen et al., 2012). This is further addressed later in the chapter.

Age-related disease presentations

For some genetic causes of epilepsy, a characteristic age of seizure onset, and sometimes offset, is seen. There are a number of studies showing that these parallel the timing of expression of the gene in the brain. Examples of this include:

 KCNQ2 and KCNQ3, genes encoding neuronal potassium channels, are expressed in late fetal life and early infancy, with expression reducing after this age. The epilepsy associated with these genes is usually on day 2-3 of life, and typically has offset by early childhood (often in mid-infancy) (Kanaumi et al., 2008; Weckhuysen et al., 2012; Zara et al., 2013).  SCN2A and SCN8A, the major genes coding for the sodium channels on excitatory neurons have different patterns of expression. SCN2A has a peak

expression in late fetal and early infant periods, decreasing after this time concurrent with an increase in SCN8A expression. The mean age of epilepsy onset in SCN2A is in the first week of life and in SCN8A is five months old (Howell et al., 2015; Larsen et al., 2015; Liao et al., 2010).  SCN1A, the gene for Dravet syndrome which has onset between four and fifteen months old is expressed at low levels neonatally, increasing sharply with age to a peak at 20 months old, from which point expression continues at approximately the same level. The authors report parallel timing in the reduction of expression of another member of the sodium channel gene family, SCN3A, with expression levels crossing at 5-6 months, which is the peak age of Dravet syndrome onset. They postulate that SCN3A expression was able to compensate for abnormal levels of SCN1A expression for a time, this being lost as SCN3A expression reduces (Cheah et al., 2013).

Potential novel and targeted therapies

With the exception of Glut-1 deficiency and the vitamin-responsive epilepsies, specific treatments are not available for genetic epilepsies (Hunt et al., 1954; Klepper et al., 2005). The development of therapies targeted to the underlying cause has been a major focus of research in recent years.

To date, the most immediately translatable advances have come in ‘repurposing’ existing drugs whose mechanisms of action are expected to remedy the molecular defects. These include targeted use of existing AEDs, such as the potassium channel opener retigabine in KCNQ2 encephalopathy, and sodium channel blockers, particularly phenytoin in SCN2A and SCN8A encephalopathies. Drugs not previously used in epilepsy have been repurposed, such as memantine for mutations of NMDA receptor genes, quinidine in KCNT1 encephalopathy and everolimus in mutations of genes in the mTOR pathway (Bearden et al., 2014; French et al., 2016; Fukuoka et al., 2016; Millichap et al., 2016; Milligan et al., 2014; Pierson et al., 2014). However, while these treatments have held promise, reported benefits in patients have been modest and improvement not universal (Chong, Nakamura, Saitsu, Matsumoto, & Kira, 2016; Mikati et al., 2015).

Other therapeutic approaches that hold promise include bioactive peptides, antisense oligonucleotides and gene therapy; these have not yet progressed to human trials in epilepsy (rev in (McTague et al., 2016)).

Finally, treatments which target particular molecular pathways rather than individual genetic causes should be investigated as each of these may treat a number of genetic causes rather than just one.

1.5.5 Aetiologies by epileptic syndrome Aetiologies of the ‘benign’ epileptic syndromes

Benign familial neonatal and infantile epilepsies: The benign familial neonatal and infantile epilepsies are largely autosomal dominant channelopathies, with four genes, KCNQ2, KCNQ3, SCN2A and PRRT2 (Berkovic et al., 2004; Biervert et al., 1998; Charlier et al., 1998; Heron et al., 2002; Heron et al., 2012; N. A. Singh et al., 1998)accounting for 90% of cases, and no mutation identified in the remaining 10% (Grinton et al., 2015; Zara et al., 2013). Very recently, a fifth gene was reported, with SCN8A mutations identified in three families with BFIE (Gardella et al., 2016).

KCNQ2 and KCNQ3 mutations have been identified in BFNE, and SCN2A (and rarely KCNQ2) mutations in BFNIE. Mutations in all five genes are described in BFIE, but approximately 70% of cases are due to PRRT2 mutations (Zara et al., 2013).

While seizure semiology and good response to treatment are common across all genotypes in these syndromes, the following differences between genotypes have been identified:

 KCNQ2 mutations predominantly have a neonatal presentation and PRRT2 mutations an infantile presentation (Deprez et al., 2009; Zara et al., 2013).  Seizures beyond infancy are seen in some patients with KCNQ2 or SCN8A mutations and some with unknown gene mutations, but rarely with SCN2A or PRRT2 mutations (Gardella et al., 2016; Grinton et al., 2015; Zara et al., 2013).

 Presence of later-onset paroxysmal kinesogenic dystonia or choreoathetosis in patients with PRRT2 and SCN8A mutations (Gardella et al., 2016; Heron et al., 2012).

Benign myoclonic epilepsy of infancy: The genetic basis of BMEI is unknown, although a 2:1 male predominance and a family history of other epileptic syndromes in one-third of patients suggests a genetic basis (Auvin et al., 2006).

Aetiologies of the ‘severe’ epileptic syndromes

There are many reported aetiologies in infants with the more severe epileptic syndromes of infancy; all aetiologies are considered in the sections below.

Early infantile epileptic encephalopathy (Ohtahara syndrome): Major developmental structural abnormalities are reported in EIEE, with a range of both malformations of cortical development including diffuse abnormalities such as lissencephaly, bilaterally asymmetric conditions such as Aicardi syndromes, and unilateral abnormalities like hemimegalencephaly. A number of infants with ‘cerebral dysgenesis’ causing EIEE are reported; further detail is not available to determine the nature of the imaging findings (Yamatogi & Ohtahara, 2002) Hindbrain malformations such as pontocerebellar hypoplasia are also described (Saitsu et al., 2012). One of the largest series of EIEE reported structural aetiologies in 50% of infants; the rate is lower in other studies (Clarke et al., 1987; Yamatogi & Ohtahara, 2002).

The remaining patients have normal or non-specifically abnormal brain imaging and in recent years have had ‘genetic’ causes identified. Although no population-based studies have been conducted, four genes have been reported to account for 5-30% of cases of non-lesional EIEE (in different series). In decreasing order of frequency, these are STXBP1, KCNQ2, SCN2A and GNAO1. All four are autosomal dominant conditions, typically due to de novo mutations, although inheritance from a mosaic parent is reported in three of these genes (Howell et al., 2015; Kim et al., 2013; Nakamura, Kato, et al., 2013; Nakamura, Kodera, et al., 2013; Pisano et al., 2015; Saitsu et al., 2016; Saitsu et al., 2011; Saitsu et al., 2008; Stamberger et al., 2016; Weckhuysen et al., 2013; Weckhuysen et al., 2012; Zerem et al., 2014).

Features which distinguish these four genetic causes of EIEE are:

 Epilepsy onset in KCNQ2 is almost universally in the first week of life, with a broader range of onset in the other genes.  Evolution to infantile spasms is not common with KCNQ2 mutations, but is frequent with the other genes.  Seizure outcome is variable. Seizures settle in most patients with KCNQ2 mutations by age 3, but are ongoing and refractory in most patients with SCN2A and GNAO1 mutations. Seizures may settle before recurring in STXBP1, with approximately half ultimately refractory.  A poor response is seen to most treatments for all genes. However, improvement in seizure control is seen with sodium channel blockers in some patients with SCN2A and KCNQ2 mutations.  Hyperkinetic movement disorders are present in most infants with STXBP1, SCN2A and GNAO1 mutations, and some with KCNQ2 mutations. While the types of movement disorders are often ‘mixed’ (i.e. more than one type of movement disorder may occur), the predominant type varies between genes. In those with STXBP1 mutations, stereotypies/tremor predominates, the movements being described as ‘figure of eight head stereotypies’ or ‘generalised tremor of head and all limbs’. In GNAO1, most infants have chorea. Dystonia, chorea and dyskinesias can all be seen in SCN2A. Where present, the movement disorders in KCNQ2 are typically less problematic than in the other genes; myokymia and dystonia are reported.

Other single gene causes that account for an unknown proportion of EIEE include ARX, BRAT1, GABRA1, SLC25A22 and QARS, as well as rare presentations of a small number of metabolic conditions such as biotinidase deficiency, CPT deficiency, non- ketotic hyperglycinaemia, pyridoxine-related and mitochondrial conditions. An unknown proportion of patients remain with unknown, presumed genetic, cause (Fusco, Pachatz, Di Capua, & Vigevano, 2001; M. Kato et al., 2007; Kodera et al., 2016; Molinari, 2010; Molinari et al., 2009; Pearl, 2016; Saitsu et al., 2014; Singhi & Ray, 2011; X. Zhang et al., 2014).

Early myoclonic encephalopathy: The cause is unknown in many patients with EME, although the proportion of patients with an identified cause is difficult to estimate given the rarity of this condition.

The majority of the reported causes of EME are metabolic, the most well described being non-ketotic hyperglycinaemia and disorders of pyridoxine metabolism related to mutations in the ALDH7A1 and PNPO genes (P. Baxter, 2001; Hoover-Fong et al., 2004; Markand, Garg, & Brandt, 1982; Mills et al., 2014; Mills et al., 2010; Pearl, 2016). Disorders of pyridoxine metabolism are critical to identify, as specific treatments are available (Mills et al., 2014; Mills et al., 2010).

Other metabolic conditions reported in small series or single cases to cause EME include molybdenum cofactor deficiency, sulfite oxidase deficiency, disorders of amino acid metabolism (D-glyceric acidaemia, propionic aciduria, methylmalonic acidaemia), Menkes disease and Zellweger disease (Pearl, 2016; Vigevano & Bartuli, 2002).

In recent years, five genes not typically associated with abnormalities of biochemical testing have been identified in infants with EME. Two, ErbB4 and GABRB2, are each reported only in a single patient (Backx EJHG 2009, Ishii JMG 2016). Three, PIGA, SIK1 and SLC25A22 have each been reported in a small number of unrelated patients (Cohen et al., 2014; Hansen et al., 2015; Johnston et al., 2012; M. Kato et al., 2014; Molinari et al., 2009; Molinari et al., 2005). Each of these genes has a different role in neuronal function, without obvious overlap to suggest a common mechanism of cellular dysfunction. Clinical features that suggest particular genes are not yet clear, given the small number of reported cases. However, dysmorphism is reported with PIGA and SLC25A22 mutations, and infants with PIGA mutations may have contractures and an elevated serum alkaline phosphatase level.

Currently, the diagnostic work-up of EME focuses on the biochemical testing for metabolic conditions given these are the most well-described causes of EME, and that infants with a disorder of pyridoxine metabolism may have better outcomes if treatment is instituted early. The aforementioned metabolic conditions are typically all tested for

as the clinical features of these conditions in the neonatal period are often non-specific and overlapping.

Epilepsy of infancy with migrating focal seizures: In 2011, KCNT1 was identified as the causative gene in 50% of a group of 12 patients with EIMFS, indicating that it is likely the major cause of this condition. Subsequent work has demonstrated significant genetic heterogeneity. SCN2A mutations account for approximately 25% of cases, and SCN1A mutations or deletions are reported in 14% of patients in whom SCN1A testing is commented on. It is certainly worth noting though, that EIMFS is a rare phenotype among those with SCN1A mutations (Barcia et al., 2012; Carranza Rojo et al., 2011; Harkin et al., 2007; Howell et al., 2015; McTague et al., 2013).

Phenotypic features of EIMFS that predict the genotype among these three causes are less well delineated than in EIEE, although response to sodium channel blockers and presence of a movement disorder may suggest SCN2A.

A number of other genes are reported in a small number of patients, and account for an unknown proportion of cases of EIMFS. Single gene causes include dominant mutations in CACNA1A and SCN8A and mutations in recessively inherited genes including PLCB1, QARS, SLC12A5, SLC25A22 and TBC1D24 (Epi & Epi, 2016; Milh et al., 2013; Ohba et al., 2014; Poduri, Chopra, et al., 2012; Poduri et al., 2013; Stodberg et al., 2015; X. Zhang et al., 2014).

Metabolic causes of EIMFS are reported; EIMFS may be a relatively common epileptic syndrome among infants with congenital disorders of glycosylation, occurring in 4/17 patients in a recent case series. Only a small number of case with brain malformations and one with a chromosomal abnormality (duplication 16p11.2) are reported (Barba et al., 2016; Bedoyan et al., 2010; Coppola et al., 2007; Fasulo, Saucedo, Caceres, Solis, & Caraballo, 2012).

Infantile spasms/West syndrome: The causes of infantile spasms are extremely heterogeneous, with over 200 individual aetiologies reported. It should be noted that many of these studies were conducted in groups with infantile spasms rather than the epileptic syndrome of West syndrome more specifically, therefore including those in

whom hypsarrhythmia was not present. It is also notable that, in some, infantile spasms is the first (and sometimes only) seizure type, and in others, infantile spasms are preceded by other seizure types; many studies of infantile spasms aetiology do not separate these groups. It is not clear whether the aetiologies of West syndrome are different to those of all infantile spasms, nor whether the aetiologies in those with spasms at epilepsy onset are different from those with spasms beginning after an evolution of the epilepsy. The significance or otherwise of these distinctions is not further considered here (Lux & Osborne, 2004; Matsumoto et al., 1981; Okumura et al., 1998; Riikonen, 1982).

Two large recent studies of infantile spasms identified structural brain disorders, including both developmental brain abnormalities and acquired brain insults in approximately 40% of patients (Knupp et al., 2016; Osborne et al., 2010). Developmental brain abnormalities associated with infantile spasms include many malformations of cortical development, as well as malformations considered predominantly hindbrain malformations (Parrini et al., 2016). The former include lissencephaly related to LIS1, DCX, TUBA1A and ARX genes, TS, FCDs and hemimegalencephaly related to mutations in genes in the mTOR pathway (e.g. TSC1, TSC2, DEPDC5, NPRL2, NPRL3 and MTOR), PMG, other neurocutaneous syndromes including neurofibromatosis type 1 and hypomelanosis of Ito, and Aicardi syndrome (Aicardi, 2005; Crino, 2015; Parrini et al., 2016; Pascual-Castroviejo et al., 1998). The latter include many of the pontocerebellar hypoplasias, including those associated with the CASK and RARS2 genes (Cassandrini et al., 2013; Michaud et al., 2014). A number of studies report that some clinical features suggest a structural (usually focal or asymmetric bilateral) aetiology, namely asymmetric spasms, focal seizures in addition to spasms, hemihysarrhythmia and asymmetric ictal EEG (Donat & Lo, 1994; E. K. Gaily et al., 1995; Kramer, Sue, & Mikati, 1997).

Approximately 8% of patients with infantile spasms have a chromosomal abnormality (Osborne et al., 2010). West syndrome is well-described in six syndromes, including Down syndrome (trisomy 21), Williams syndrome (del 7q11.23), Miller-Dieker syndrome (del 17p13.3), Pallister-Killian syndrome (tetrasomy 12p), del 1p36 and maternal dup 15q11q13. West syndrome is also reported as an infrequent feature in

many other chromosomal anomalies, including del 4p, del 4q35, ring chromosome 9, dup 16p11.2, del 17p11.2, del 19p13.13 and dup 21q21 (A. Battaglia & Guerrini, 2005; R. Singh, Gardner, Crossland, Scheffer, & Berkovic, 2002).

Metabolic conditions make up 1-7% of cases in different series of infantile spasms. Infantile spasms has been reported in many metabolic conditions, although it is a well- recognised feature of only a small number: ‘Developmental delay, epilepsy and neonatal diabetes syndrome’, phenylketonuria, non-ketotic hyperglycinaemia, methylmalonic acidaemia, propionic acidaemia, maple syrup urine disease and Menkes disease. IS can be seen, albeit less frequently, in pyridoxine-related epilepsies, mitochondrial conditions including Leigh’s disease and pyruvate dehydrogenase deficiency, and congenital disorders of glycosylation. It is important to note that, in some metabolic aetiologies of infantile spasms including pyridoxine-dependent epilepsies , there are no reports of IS in patients who have not had other seizure types prior to spasm onset (Bahi-Buisson et al., 2007; Bahi-Buisson et al., 2006; Dalla Bernardina, Aicardi, Goutieres, & Plouin, 1979; Gkampeta & Pavlou, 2012; Liu et al., 2015; Matsumoto et al., 1981; Mills et al., 2010; Morava et al., 2016; Osborne et al., 2010; Patel, O'Brien, Subramony, Shuster, & Stacpoole, 2012; Pearl, 2016; Tsuji et al., 2003; Verrotti et al., 2014; Vigevano & Bartuli, 2002; Wirrell et al., 2015; Zhongshu, Weiming, Yukio, Cheng, & Zhixing, 2001).

Many single gene disorders causing infantile spasms have been identified in recent years, and are shown in Figure 1.2. However, the relative frequency of each ‘genetic’ cause at a population-level, and the proportion in which a genetic diagnosis can now be achieved, is not clear. Mutations in CDKL5 and STXBP1 are the two best known ‘genetic’ causes of infantile spasms, although in almost all cases, other seizures (and developmental delay) predate the onset of spasms (Bahi-Buisson, Kaminska, et al., 2008; McTague et al., 2016; Stamberger et al., 2016).

The genetic heterogeneity of infantile spasms has led to the idea that common pathways or mechanisms are disrupted at the right age to produce disease. Bioinformatic studies indicate that genes that cause infantile spasms likely have a role in both forebrain and interneuron development (Paciorkowski et al., 2011). The implicated genes may

converge on particular biologic pathways, with mutations in a number of subunits of the GABA receptor identified, and studies of protein-protein interaction demonstrating a number of other critical networks (Consortium et al., 2013). Further work into these pathways may elucidate the mechanisms by which West syndrome arises, and may also suggest other candidate genes (Oliver et al., 2016; Oliver et al., 2014).

With current diagnostic strategies, the cause is unknown in approximately one-third of patients despite investigation (Osborne et al., 2010). These patients have a presumed genetic basis for their epilepsy.

Dravet syndrome: Dravet syndrome is probably the most genetically homogeneous of the epileptic (and genetically explained) syndromes, with more than 80% of patients having a mutation in the SCN1A gene. 90% of mutations arise de novo, and 10% are inherited, either from a parent with a personal or family history of GEFS+, or from a parent with somatic mosaicism (Brunklaus et al., 2012; Depienne, Trouillard, et al., 2009; Xu et al., 2015). Approximately 85% of patients with Dravet syndrome have a sequencing mutation in SCN1A, and copy number variants are identified in 3-5% (refs). Approximately 50% of the reported mutations are missense, occurring predominantly in the conserved voltage sensor and ion pore regions of the gene, and 50% are truncation mutations (Brunklaus et al., 2012; Claes et al., 2001; Depienne, Trouillard, et al., 2009; Djemie et al., 2016; Suls et al., 2006; Xu et al., 2015).

Mutations in the X-chromosome gene, PCDH19, are also reported as a cause of Dravet syndrome in female SCN1A-negative patients. However, while there are some similarities between the epilepsy associated with PCDH19 mutations and SCN1A- associated Dravet syndrome, in most cases these can be distinguished on clinical grounds (Depienne, Bouteiller, et al., 2009; Trivisano et al., 2016).

More recently, a ‘Dravet-like’ phenotype has been reported in patients with CHD2 mutations given the presence of both febrile and afebrile seizures, multiple seizure types including myoclonic seizures starting in the second year of life or later, normal or near normal development before epilepsy onset and later intellectual disability. However, these patients are clearly distinct from those with Dravet syndrome due to their later age

of epilepsy onset (14-42 months), as Dravet syndrome almost always begins before 15 months (Suls et al., 2013; Thomas et al., 2015).

Finally, Dravet- or Dravet-like syndromes have been reported in patients with HCN1, KCNA2, GABRA1, GABRG2, SCN1B and STXBP1 mutations. As yet, only a small number of cases are reported for each gene; it remains to be seen whether features distinct from typical Dravet syndrome will become apparent (Carvill et al., 2014; Kang & Macdonald, 2016; Nava et al., 2014; Patino et al., 2009; Syrbe et al., 2015).

Myoclonic encephalopathy in a non-progressive disorder: Myoclonic encephalopathy in a non-progressive disorder (MENPD) is associated with severe acquired brain insults, particularly hypoxic-ischaemic encephalopathy, and genetic disorders, most notably Angelman syndrome. In Angelman syndrome, MENPD has been reported in 30-50% with 15q11q13 deletions and UBE3A mutations, but does not appear common in patients with uniparental disomy. Other reported aetiologies include Prader-Willi and Wolf-Hirschhorn syndrome and MECP2 mutations, and malformations such as polymicrogyria (R. H. Caraballo et al., 2007; Elia, 2009).

‘Structural’ or ‘symptomatic’ focal epilepsies:

The aetiologies associated with these epilepsies largely overlap with the structural and metabolic aetiologies of West syndrome, with both epilepsy phenotypes having been reported in many of these conditions.

The literature suggests that focal and multifocal structural abnormalities can present with either ‘structural focal epilepsy or West syndrome, where as those with more diffuse structural abnormalities more commonly present with West syndrome. Evidence of this is seen in series that focus on a single underlying aetiology. In infant-onset epilepsy associated with FCD, the first seizure type was spasms in 14-33%, with a larger proportion of the group having focal seizures (Fauser et al., 2006; Lortie, Plouin, Chiron, Delalande, & Dulac, 2002). Studies of more diffuse aetiologies, neonatal HIE, periventricular leukomalacia and lissencephaly report that, while focal seizures are seen in a small number, infantile spasms are the main seizure type in infancy (Bittar, Rosenfeld, Klug, Hopkins, & Harvey, 2002; M. Duchowny et al., 1998; Herbst et al.,

2016; T. Kato et al., 2010; Maton et al., 2008; Okumura, Hayakawa, Kuno, & Watanabe, 1996; Watanabe et al., 1980).

The major causes of symptomatic focal epilepsies with onset in infancy reported in epilepsy surgery series are FCD including hemimegalencephaly, glioneuronal tumours, TS, Sturge Weber syndrome and perinatal stroke (M. S. Duchowny et al., 1990; Jonas et al., 2004; Loddenkemper et al., 2007; Wyllie et al., 1996).

Aetiologies of the ‘complex’ epileptic syndromes

A growing number of aetiologies is reported to cause severe infantile epilepsies that do not fit a well-described epileptic syndrome. The majority of associated aetiologies have been reported in only a small number of patients to date and the descriptions of the epilepsy are often minimal. The genes associated with the particular ‘subgroups’ noted in the Epileptic features section above are shown in Figure 1.2.

The ‘genetic’ causes of ‘severe’ infant epileptic syndromes, adapted from McTague and Howell et al 2016, are shown by syndrome in Figure 1.2 below (McTague et al., 2016).

Figure 1.2 Genetic aetiologies of the ‘severe’ infant epileptic syndromes

Figure adapted from McTague et al1. Genetic causes of severe infant epileptic syndromes. Only non- chromosomal, non-malformative and non-metabolic conditions are included. Genes reported in just one patient or family, and those predominantly associated with later-onset epilepsies, are not listed. Black font denotes genes that account for >50% of cases of that syndrome, purple font 10- 50%, blue font <5%, and green font an unknown percentage of cases.

1 Adapted from Figure 1 in McTague and Howell et al, Lancet Neurology 2016. I designed that figure, as noted in the Contributors section of that paper.

1.5.6 Aetiology by non-epileptic features Distinguishing the aetiologies of SEI is sometimes aided by the presence of specific, non-epileptic clinical features. Non-neurologic features are often more useful; neurologic features are often less unique given developmental delay, tone abnormalities and movement disorders are seen in many aetiologies. Examples of aetiologies associated with particular clinical features include:

 hearing impairment in TBC1D24, KCNJ10, SETBP1 and SPATA5 mutations, metabolic aetiologies such as RFT1-associated congenital disorder of glycosylation, some mitochondrial disorders, and acquired causes such as congenital cytomegalovirus and meningitis (Bockenhauer et al., 2009; Campeau et al., 2014; Goderis et al., 2014; Hoischen et al., 2010; Rodenburg-Vlot, Ruytjens, Oostenbrink, Goedegebure, & van der Schroeff, 2016; Tanaka et al., 2015)  abnormal teeth seen in PIGA, ROGDI and SLC13A5 mutations  skeletal abnormalities including skeletal dysplasia associated with FGFR3 (achondroplasia, hypochondroplasia, Muenke syndrome) and PEX7 (rhizomelia chondrodysplasia punctate) genes, frequent fractures with PURA mutations, and triphalangeal thumb +/- large or absent distal digits in TBC1D24 (Braverman, Moser, & Steinberg, 1993; Campeau et al., 2014; Lalani et al., 2014; Linnankivi, Makitie, Valanne, & Toiviainen-Salo, 2012) Non-epileptic clinical and imaging features associated with genetic (non- malformative, non-metabolic, non-chromosomal) aetiologies of SEI are listed in Appendix A.

1.5.7 Infantile epilepsies of unknown aetiology The cause remains unknown in approximately half of infants with epilepsy, and approximately one-third of patients with IS (Eltze et al., 2013; Osborne et al., 2010). These patients are presumed to have a genetic basis for their epilepsy, the evidence for which includes that no alternative aetiology has been identified despite extensive investigation, reports of multiple affected family members in some cases and a gender bias in some phenotypes (Auvin et al., 2006; E. Gaily et al., 2016; Wirrell et al., 2015).

The presumption has been proven in an increasing number of instances. Many genes for infantile epilepsy have now been identified in patients with previously unknown cause. This is further discussed later in the chapter.

1.5.8 Implications of identifying aetiology Making an aetiologic diagnosis has implications for identifying the most appropriate therapies in an individual patient, minimising unnecessary, invasive and expensive diagnostic investigations, informing prognosis and, in the case of genetic aetiologies, providing specific and accurate reproductive counselling to families of affected infants.

Dravet syndrome is a good example of the ways in which genetic testing can improve patient care and outcomes even in the absence of specific treatment for the condition. A large survey of parents and physicians caring for children with Dravet syndrome found positive genetic testing to be helpful in most patients (87% per parent report), preventing additional investigations, altering treatment approaches and access to therapies and, in 42% (per physician report), improving seizure control through medication change (Brunklaus et al., 2013).

In the future, it is hoped that the knowledge gained from identifying new aetiologies for infantile epilepsies will advance knowledge of the mechanisms of epileptogenesis in the developing brain, and guide development of novel and targeted therapies.

1.6 Investigating aetiology of ‘severe’ epilepsies of infancy

1.6.1 Current approach The approach to investigation of aetiology in infantile epilepsies differs between those with ‘benign’ and ‘severe’ epilepsies.

In the ‘benign’ epilepsies, if there is a family history of neonatal or infantile seizures, the infant may not undergo investigation, other than genetic testing. If there is no family history, the infant typically has brain imaging performed. Other investigations such as chromosomal testing and some basic metabolic tests on blood and urine may be performed. More invasive or expensive metabolic testing is typically not undertaken

unless additional clinical features are suggestive, as well-controlled epilepsy in infancy, in association with normal development, is rarely due to metabolic aetiologies.

The current approach to aetiologic diagnosis in the ‘severe’ epilepsies of infancy depends on whether clinical assessment suggests a specific cause. Where this is the case, testing is targeted to the presumed diagnosis. Examples include:

 Brain MRI in infants with depigmented patches suggesting TS  Brain MRI in infants with hemiplegia suggesting perinatal stroke or a brain malformation  Chromosomal or single gene testing in infants with particular dysmorphic syndromes  SCN1A testing in infants with Dravet syndrome

Often though, a specific diagnosis is not suspected following clinical assessment, and the patient undergoes a non-targeted diagnostic work-up that typically occurs in a tiered or staged fashion, with tests prioritized that are potentially management-changing, more likely to yield a result, less expensive and less invasive. The specific testing done depends on what is available, and varies from institution to institution, but typically includes imaging, chromosomal testing and often extensive metabolic testing (Osborne et al., 2010; Wirrell et al., 2015). The initial investigations are typically done at presentation, and repeat imaging and more detailed metabolic testing is often performed at later time points if no diagnosis is reached. Currently in Victoria, single gene testing is performed only when a specific cause is thought highly likely and a genetic diagnosis is management-changing (e.g. SCN1A in Dravet syndrome) and next generation genetic testing is not available on a clinical basis due to funding constraints despite recently becoming available commercially.

There is little information on the diagnostic yield of each investigation in infants with epilepsy. MRI brain imaging is the investigation with highest yield, with an aetiologically relevant finding identified in 25-50% of infants with epilepsy onset between one and 24 months (Berg et al., 2009; Eltze et al., 2013), with approximately 50% of these infants having developmental brain abnormalities, and 50% acquired brain insults. There are a number of issues with MRI brain imaging in infants, of which

clinicians need to be aware and, where feasible, approaches to scanning and interpretation of images modified to account for these. MRI in infants with SEI needs to be designed to detect structural abnormalities, including small FCDs, as these are potentially operable. A major limitation is the changing signal of white matter during the first two years of life as postnatal myelination occurs. This reduces the contrast between grey and white matter, particularly at intermediate stages of myelination (between 8-18 months), limiting detection of FCDs, which characteristically show blurred grey-white differentiation (Vezina, 2011; Woermann & Vollmar, 2009; Yoshida et al., 2008). In fact, FCDs visible on neonatal imaging may ‘disappear’ on imaging later in infancy, and ‘reappear’ after two years old (Eltze et al., 2005). Thus, if the initial imaging did not reveal an FCD where this is clinically suspected, repeat imaging at an older age should be consider. Within the infant period though, the MRI technique can be optimized to improve detection of FCDs; studies show improved identification with 3T MRI compared with 1.5T, although this has not been looked at in infants (Knake et al., 2005; Nguyen et al., 2010). Particular MRI sequences recommended for detection of structural abnormalities in all age groups should be used, with some modifications, including the use of double inversion recovery sequences instead of FLAIR sequences, which are beneficial as they completely suppress the white matter signal and partly overcome the issue of poor grey-white contrast in this age group (Soares, Porter, Saindane, Dehkharghani, & Desai, 2016; Vezina, 2011). Finally, the importance of imaging review by an experienced neuroradiologist who has knowledge of the infant’s clinical presentation (e.g. seizure semiology, EEG findings, other clinical features including focal findings on neurologic examination) in optimizing rates of FCD detection cannot be underestimated (Woermann & Vollmar, 2009). Other imaging technologies, particularly fluorodeoxyglucose positron emission tomography (FDG- PET), are sometime useful, particularly if a focal structural abnormality is suspected. Focal areas of hypometabolism can sometimes be identified on PET in infants with FCD when MRI did not identify an abnormality (Chugani et al., 1988; Chugani et al., 1990).

Studies that have looked at yield of particular investigations in particular epileptic syndromes include the UKISS study of infantile spasms: among infants with an aetiology identified, 35% of patients had an aetiology known from clinical history or

past investigations and did not require additional investigations (e.g. history of HIE), 7% had an aetiology identified on clinical examination, 43% on neuroimaging, 13% on chromosomal testing, 2% on other testing and 1% unknown. No patient had a diagnosis made on urine metabolic screen (Osborne et al., 2010). It should be noted that the proportion of infants with an aetiologic diagnosis made on clinical history or prior investigations is likely to be lower in the other epileptic syndromes than found in the UKISS study given that many remote symptomatic (i.e. known) aetiologies present with spasms (Okumura et al., 1996; Watanabe et al., 1980; Watanabe, Kuroyanagi, Hara, & Miyazaki, 1982). Additionally, the proportion of infants with a diagnosis made using each testing modality is likely to vary between epileptic syndromes; data is not available for the other syndromes. However, for some syndromes such as EIMFS, the diagnostic yield of imaging, metabolic and chromosomal testing is minimal (Coppola, 2009; McTague et al., 2013).

The current standard, ‘tiered’ testing performed in infants with unknown cause for ‘severe’ epilepsy at RCH is as follows:

Table 1.5 Tiered investigations currently performed in infants with severe epilepsies of unknown cause

Tier Investigations

First tier MRI brain imaging (on all infants) Ophthalmology assessment Hearing test Wood’s lamp assessment Blood tests – SNP microarray (and parental testing if copy number variant identified), FBE, UEC, glucose, Ca/Mg/PI, LFTs, biotinidase, lactate, ammonia, amino acids, acylcarnitines, vitamin B12, copper and caeruloplasmin, uric acid Urine ‘metabolic screen’ – organic acids, amino acids, P6C, S- sulphocysteine, guanidinoacetic acid, purines and pyrimidines Second tier Blood – Karyotype by G-banding, mitochondrial mutations, (all to be done if first tier normal, POLG common mutations, transferrin isoforms, very long chain limited testing if clinical features fatty acids, white cell enzymes and first-tier investigations Cerebrospinal fluid (CSF) – cell count, protein, glucose*, lactate*, suggestive of mitochondrial pyruvate*, amino acids*, neurotransmitters, MTHFR (* = paired disorder) with serum) Repeat brain imaging 3T MRI brain imaging with epilepsy-specific sequences

Third tier Biopsy of skin, muscle and liver for respiratory chain enzyme (only where clinically indicated analysis, biopsy of skin for electron microscopy for changes of and requested) neuronal ceroid lipofuscinosis Other Targeted single gene testing (if particular aetiology thought highly likely)

1.6.2 Genetic investigation Over time in the epilepsy field, research genetic testing has moved from ‘gene-by-gene’ approaches guided by techniques such as linkage analysis and homozygosity mapping, to NGS approaches that sequence large numbers of genes in parallel. Such techniques, including targeted multigene panels and WES are now being used not only for gene discovery, but also as diagnostic tools. Whole genome sequencing (WGS) approaches remain largely in the research domain due to challenges of analyzing such large amounts of data, and limited knowledge of normal and disease-associated genetic variation outside the exome (rev in (McTague et al., 2016).

In the last couple of years, several groups have published their experience with the use of targeted multigene panels and WES in epilepsy, predominantly in ‘severe’ epilepsies (Table 1.6), reporting pathogenic mutations in 10-72% of patients (Allen et al., 2016; Carvill, Heavin, et al., 2013; Consortium et al., 2013; de Kovel et al., 2016; Della Mina

et al., 2015; Dimassi et al., 2016; Dyment et al., 2015; Euro, Epilepsy Phenome/Genome, & Epi, 2014; Gokben et al., 2016; Kodera et al., 2013; Lemke et al., 2012; Martin et al., 2014; Mercimek-Mahmutoglu et al., 2015; Michaud et al., 2014; Veeramah et al., 2012; Wang, Gotway, Pascual, & Park, 2014). The large differences in reported yield are not comparable as there were differences in patient groups studied with regards to phenotype and previous genetic testing.

Table 1.6 Use of next generation sequencing in epilepsy

Number Diagnostic Paper Patient population NGS technique of yield patients Lemke 2012 Mixed population with Targeted NGS 33 49% of epilepsy (including specific panel (265 genes) testing naïve syndromes such as Dravet patients syndrome) Carvill 2013 Any epileptic encephalopathy Molecular 500 10% of inversion probes previously (MIPS) (65 genes) unsolved cases Epi4K 2013 Infantile spasms or LGS WES (trio) 264 Not stated

Kodera 2013 Early onset epileptic RNA bait (35 53 23% encephalopathies genes) Veeramah 2013 Refractory seizures with WES (trio) 10 70% cognitive, behavioural or neurologic comorbidity, onset <8.5 years Michaud 2014 Infantile spasms WES (trio) 18 28%

EuroEpinomics Infantile spasms or LGS WES (trio) 356 12% 2014 Wang 2014 Children with epilepsy Targeted NGS 19 47% panel (38-53 genes) Martin 2014 Early onset epileptic WGS 6 67% encephalopathies Della Mina 2015 Epilepsy onset <4 years Targeted NGS 19 47% panel (67 genes) Dyment 2015 Early onset epileptic WES 11 72% encephalopathies, or childhood onset seizures not part of a recognized syndrome Mercimek- Refractory epilepsy and Targeted NGS 93 15% Mahmutoglu developmental delay panel (38-70 2015 genes in most, 327 genes in some) De Kovel 2016 Seizures onset <5 years, and Targeted NGS 360 8% intellectual disability panel (377 genes – NB 351 were candidate genes) Allen 2016 Infant-onset EE (all onset <2 Targeted NGS 50 22% years, 95% < 1year) panel (137 genes) Gokben 2016 Early onset epileptic Targeted NGS 30 40% encephalopathies panel (16 genes) Dimassi 2016 Infantile spasms WES (trio) 10 40%

These techniques have inherent limitations, including inability to sequence intronic regions and limited ability to sequence repeat regions, and drawbacks such as inconsistent coverage of genes, false positive results necessitating confirmation of detected variants with Sanger sequencing, and requirement (in many instances) for parental samples (for segregation analysis for determination of pathogenicity) (Precone et al., 2015). However, they have the significant advantage of being able to sequence a large number of genes in short time at relatively low cost. Currently, the cost of targeted multigene approaches is estimated to be the same as sequencing 1-3 medium-large genes by conventional techniques (Lemke et al., 2012). Saitsu et al reported being able to perform targeted multigene panel testing on 24 patients in one week (Kodera et al., 2013).

Just a single small study has looked at the yield of WGS in epilepsy, finding pathogenic mutations in four of six infants with early onset epileptic encephalopathies (Martin et al., 2014); it will likely be some time before this technique is in clinical use.

1.6.3 Future of diagnostic investigation Although it is likely that most of the infants with epilepsy of unknown aetiology have a genetic basis for their epilepsy, genetic testing is not currently widely available outside of a research setting.

It is expected that, in the future, genetic testing will be a routine part of the diagnostic process, most likely performed early. This is already happening at some centres (Helbig et al., 2016). It is not clear yet what type of testing (e.g. single gene, gene panel, WES, WGS etc.) will be the most efficient and cost-effective for diagnosis in this group of patients, and whether this will vary between epileptic syndromes. It seems likely though, given the genetic heterogeneity of the infantile epilepsies, that a multigene approach is likely to be superior to a single gene approach for most situations.

1.6.4 Cost of diagnostic investigation The cost of diagnostic investigation in patients with infantile epilepsy has not been systematically studied.

Estimates suggest that approximately 25-50% of infants have an aetiology (developmental or acquired brain lesion) detected on MR imaging (Berg et al., 2009; Eltze et al., 2013). Given imaging is one of the first investigations performed in most patients, the diagnostic costs in the group with aetiologically-relevant MRI findings is relatively small (although some will get follow-up genetic testing, which will increase the cost).

The diagnostic costs are also fairly low for the small proportion that has directed testing performed when a diagnosis is suggested clinically. For most of the remaining infants who undergo non-targeted testing, the diagnostic costs are very high, and a diagnosis is often not reached despite this extensive and expensive testing. The costs of specific tests are provided in the Methods chapter.

1.7 Epidemiology of infantile epilepsies

1.7.1 Considerations in interpretation of epidemiologic studies of epilepsy This section has been left until late in this literature review, as data on incidence of epileptic syndromes and aetiologies are more easily understood and contextualized after the reader is familiar with aspects of infantile epilepsies.

When reviewing epidemiologic data on infantile epilepsies, one must consider methodologic factors that impact data quality.

Considerations in any epidemiologic study of epilepsy

It is important to appreciate that epilepsy is not a single condition, rather a group of conditions that feature recurrent, unprovoked seizures. These heterogeneous conditions vary with respect to factors such as aetiology and prognosis, and such differences limit comparability of data within and between populations.

There is considerable variability in the quality of epidemiologic studies of epilepsy. Potential issues include completeness of case identification, collection of sufficient clinical detail to maximize accuracy of diagnosis and classification, and non-standard use of terminology and measurement indices (rev in (Thurman et al., 2011)).

Recommended standards have been developed for epidemiologic studies of epilepsy to aid interpretation of reported figures, and maximize comparability of data between studies (Thurman, 1993). Where possible, study design should be population-based and prospective to maximize representativeness of data. Data collection methods should be economic, acceptable to subjects, and accurate. In studies of epilepsy, multiple sources of data, including population survey and examination, medical records from hospitals, clinics and EEG laboratories, and existing coded data, are typically preferable to single data sources to maximize accuracy of diagnosis and classification. Standard definitions exist for epilepsy, and for epileptic seizures that are not considered synonymous with epilepsy, such as FS. Current standards should be used for classification of seizure type, syndrome and aetiology, although revisions to classification systems limit comparison with older studies. Standard measurement indices should be used to report the frequency of epilepsy in a population. The specific measures chosen depend on the study type. Common measures include point prevalence and incidence rate. The point prevalence is the proportion of individuals in a population affected by a health condition at a single point in time, typically expressed as number affected per 1000 population, and is useful for determining the burden of disease in a population. The incidence rate is the frequency with which new cases occur in a population, typically expressed per 100,000 population per year. Incidence-based studies are superior to prevalence-based studies for investigation of aetiology.

Problems with studies of infantile seizures and epilepsy

Studies of the epidemiology of epilepsy are often conducted across a range of ages, with variability in the age groups for which data are reported (e.g. 0-1 year old, 0-5 years old, 0-9 years old). As such, some epidemiologic studies that include infants may not report rates of epilepsy in infancy (P. Camfield & Camfield, 2015b; Hauser, Annegers, & Kurland, 1993).

Further, standard epidemiologic definitions of epilepsy contain specific exclusions considered not to represent epilepsy proper, including neonatal seizures, FS, provoked seizures and single seizures (Fisher et al., 2014; Fisher et al., 2005). The incidence of all seizures in infancy is considerably higher than the incidence of infantile epilepsy. This is partly accounted for by FS, which are significantly more common than infantile

epilepsy, having a cumulative incidence of 2-6% in European and American studies (Nelson & Ellenberg, 1976; Verity et al., 1985a). The exclusion of neonatal seizures from earlier epilepsy definitions mean, in some studies (Fisher et al., 2005; ILAE, 1989), that epilepsies with onset in the first month of life are not considered as epilepsy for epidemiologic purposes, and therefore the entire spectrum of infantile-onset epilepsies are not considered together.

1.7.2 Incidence of infantile epilepsies Studies in multiple populations around the world identify the incidence of epilepsy to be highest in the first year of life, with figures ranging from 87-158/100,000/year (some studies excluding neonates) (C. S. Camfield, Camfield, Gordon, Wirrell, & Dooley, 1996; Casetta et al., 2012; Dura-Trave, Yoldi-Petri, & Gallinas-Victoriano, 2008; Eltze et al., 2013; C. M. Freitag, May, Pfafflin, Konig, & Rating, 2001; E. Gaily et al., 2016; Kurtz, Tookey, & Ross, 1998; Olafsson et al., 2005; Rantala & Ingalsuo, 1999; Wirrell, Grossardt, Wong-Kisiel, & Nickels, 2011). Incidence rates reduce from the second year of life, with incidences reported to range from 23.4-150/100,000/year (C. S. Camfield et al., 1996; Eltze et al., 2013; Olafsson et al., 2005), steadily declining across childhood into mid-adulthood, and rising again in the elderly (Hauser et al., 1993).

Two studies of all epilepsy in the first year of life reported a peak age of onset of 5-6 months and 5-7 months, and a higher incidence in the first six months than the second (Chevrie & Aicardi, 1977; E. Gaily et al., 2016).Long-term epidemiologic studies have suggested a mild reduction in the incidence of epilepsy in infants over time, although the reasons for this are unclear (Hauser & Kurland, 1975; Sander & Shorvon, 1996)

Incidence of epileptic syndromes

West syndrome is estimated to make up approximately half of epilepsies presenting between 1-12 months of age (range 18-68%) (Kramer, 1999; Wirrell et al., 2011). The incidence of infantile spasms has been studied in many countries, and ranges from 6- 45/100,000/year, with most population-based studies estimating incidences of 25- 42/100,000/year (rev in (Cowan & Hudson, 1991)).

The incidence of Dravet syndrome was initially reported to be less than 1:40,000, but two more recent studies have estimated annual incidences of 1:18,000-22,000 (Bayat, Hjalgrim, & Moller, 2015; Hurst, 1990; Wu et al., 2015). The reasons for the discrepancy in figures is likely multifactorial; numbers in the earlier study were very low and the more recent studies are more truly ‘population-based’. Dravet syndrome is also far more well-recognised clinical entity now than at the time of the earlier study.

EIMFS is thought to be very rare, two studies estimating an annual incidence of 0.26- 0.55/100,000 and 1.6/100,000, although it should be kept in mind that one study was not truly population-based, and the other had just two infants with EIMFS (E. Gaily et al., 2016; McTague et al., 2013).

‘Benign’ familial and non-familial epilepsies, with onset 3-11 months, normal EEG and development, no spasms, myoclonic seizures or status epilepticus and seizure freedom by 18 months old, were recently reported to occur in 22/100,000 infants, being approximately half as common as the incidence of infantile spasms in that study (E. Gaily et al., 2016).

The incidence of other infantile epileptic syndromes is not well known. In some syndromes, population-based studies have been undertaken but figures are likely underestimates given difficulties in syndrome recognition, in other syndromes figures have been estimated from non-population-based studies such as clinic- or hospital-based cohorts. For some syndromes, the incidence is not known, but estimates have been made of their relative incidence. Reported examples include that EIEE is estimated to be ≤ 1/40 as common as West syndrome (Ohtahara & Yamatogi, 2003), and that BMEI makes up 1.3-1.72% of epilepsies presenting under one year of age (R. Caraballo et al., 1997)

A recent population-based study of epilepsy presenting before age two years found no identifiable epileptic syndrome in 58% of patients. An earlier hospital-based study of ‘cryptogenic/idiopathic’ epilepsies in the first year of life found that an established epileptic syndrome could not be allocated in 25%. These findings indicate that ‘complex’ phenotypes are common in infancy (Eltze et al., 2013; Sarisjulis et al., 2000).

Incidence of ‘severe’ epilepsies of infancy

There are very few studies of the incidence of ‘severe ‘epilepsies of infancy other than West syndrome, due in part to an absence of consistent terminology as to what constitutes ‘severe’. Such studies have used variable terminology such as ‘catastrophic epilepsies’ and ‘infantile epileptic encephalopathies’, the definitions focusing variably on presence of treatment-resistant epilepsy, developmental impairment (or high likelihood of) or a well-defined electroclinical syndrome with high likelihood of poor outcome. This issue will be discussed further later in the chapter.

Two retrospective hospital or clinic-based reviews of medical records were conducted in Japan and India on patients with ‘infantile epileptic encephalopathies’. In both studies, however, ‘infantile epileptic encephalopathies’ was considered only as having one of a list of specific epileptic syndromes (such as West syndrome or EIEE). Given this definition did not include infants who had an encephalopathic course but whose epilepsy did not fit a specific electroclinical syndrome, these studies did not include the full spectrum of severe infantile epilepsies (Hino-Fukuyo, Haginoya, Iinuma, Uematsu, & Tsuchiya, 2009; Kalra, Gulati, Pandey, & Menon, 2001).

A large study of ‘catastrophic epilepsies’ in young children, while not truly epidemiologically-based, provides useful insights into the burden of severe epilepsy in young children. Of 314 children with epilepsy onset under six years old presenting to major paediatric epilepsy centres, with greater than 10 seizures per month refractory to all medical treatments, 78% had onset under one year old, and another 10% between 1-2 years old, suggesting that the major burden of severe childhood epilepsy is in infancy (Oguni et al., 2013).

Two recent population-based studies of all infantile epilepsies found that only 16% and 46% of patients had a ‘benign’ epilepsy and normal neurodevelopmental outcome (Eltze et al., 2013; E. Gaily et al., 2016). These studies suggests that more than half of infantile epilepsy is ‘severe’ from a seizure and/or developmental point of view. Future study into the epidemiology of this group of infantile epilepsies would be aided by a consistent definition of ‘severe’ epilepsy.

Incidence by aetiology

Infantile epilepsies are extremely heterogeneous. Given this large number, epidemiologic studies usually report causes by groups rather than individual aetiologies.

Over time, studies of aetiology have changed in two main ways that limit comparisons between studies and populations.

Firstly, over time, the commonly used, and ILAE standard, aetiologic groupings have changed, leading to some difficulties in comparisons between studies. The main groupings used over time include the older ‘congenital vs acquired’ and ‘prenatal vs perinatal vs postnatal’, the ILAE standard between 1989 and 2009 of ‘symptomatic vs idiopathic vs cryptogenic’, and the current ILAE classification, introduced in 2010, of ‘structural-metabolic vs genetic vs unknown’ (Berg et al., 2010; ILAE, 1981, 1989). Regardless of the grouping used though, some information on ‘subgroups’ or specific aetiologies is often available within papers on aetiologies, usually sufficient to determine the proportion with unknown vs known cause and, of the latter, the proportion with acquired vs genetic/presumed genetic causes.

The second issue is that of changes in diagnostic technologies over time. The diagnostic yield has improved with advances in imaging techniques, particularly MRI, enabling detection of more subtle structural brain malformations (Knake et al., 2005). Structural brain abnormalities are the most common group of aetiologies in three recent population-based studies (Eltze et al., 2013; E. Gaily et al., 2016; Wirrell et al., 2011).

Currently, no cause is identified in approximately half of infants. Despite recent identification of genetic aetiologies and the emergence of genomic testing in clinical practice, which is expected to significantly increase diagnostic yield, there is no data on the epidemiology of genetic infantile epilepsies. To date, no study on the genetic basis of infantile epilepsy has had an epidemiologic design and no population-based study of infant epilepsy has undertaken genetic testing systematically.

1.8 Burden of infantile epilepsies

Infant epilepsies exert a substantial psychosocial and financial burden on families, hospital systems and communities, although much of this is difficult to quantify.

Clinicians will be familiar with the enormous psychosocial burden to those with a family affected by infantile epilepsy. Aspects of this burden include grief and stress in parents and siblings, fear of loss of the child, marriage break down and loss of parental career and other life opportunities. A number of studies report reduced quality of life in parents, with greater impacts in families of children with more severe conditions. These mainly studied the burden of childhood, not infantile, epilepsy (Beghi, Frigeni, Beghi, De Compadri, & Garattini, 2005; C. Camfield, Breau, & Camfield, 2001; Cianchetti et al., 2015; Hoare & Russell, 1995).

The financial costs of epilepsy across all age groups to the community are massive, estimated to be $12.5 billion USD in the United States in 1995 and €13.8 billion across Europe in 2010 (Begley et al., 2000; Gustavsson et al., 2011). Costs related to epilepsy include those considered ‘direct’ costs such as hospital admissions, outpatient appointments, diagnostic investigations and medication costs, as well as indirect costs such as transport and education costs, and loss of productivity in caregivers. Indirect costs are more difficult to quantify, and often omitted from study, although make up the bulk of the overall cost.

The costs of epilepsy have been studied in paediatric populations, but not specifically in infants (Argumosa & Herranz, 2000; Cramer et al., 2014; Guerrini et al., 2001; Hunter et al., 2015; Riechmann et al., 2015). These studies, showed that the highest costs are consistently found in those with drug-resistant epilepsy, and that neurologic comorbidities significantly increase cost. Thus, the costs of epilepsy in the infant population is expected to be high. Two large-scale studies of inpatient healthcare costs in paediatric hospitals in the US highlight the major public health cost of both seizures and neurologic impairment (Berry et al., 2012; Standridge & Horn, 2012), and hint at a likely disproportionate burden of cost among infants compared with older children. In 2006, children with neurologic impairments made up 24.7% of bed days and 29% of hospital charges in admissions to children’s hospitals. Over 50% of all admissions in

patients with neurologic impairments occurred in children with epilepsy. Further, the majority of all hospital admissions were in those under one year.

Specific study of infants is necessary to confirm the impression that epilepsy-related costs are high in this group, and to allow comparison of cost-effectiveness and cost- savings of future diagnostic and therapeutic strategies.

1.9 What are the severe epilepies of infancy?

Epilepsies in infancy have huge variability in severity, with some infants having treatment-responsive age-limited epilepsies with normal development and others having refractory seizures and significant developmental impairments. While many infants with seizures can be recognised by clinicians to have a more ‘severe’ epilepsy, there is currently no consensus as to how ‘severe’ is defined, and no term or definition is consistently used or accepted to refer to this group of patients.

1.9.1 Terms used to denote ‘severe’ epilepsies Terms that have been used in the literature to encompass this group of patients are ‘epileptic encephalopathy’, ‘catastrophic epilepsy’ and ‘refractory (or pharmacoresistant/intractable) epilepsy’. The aforementioned terms are variably defined and/or used in the literature (Berg et al., 2010; Berg & Kelly, 2006; Kwan & Brodie, 2010; Oguni et al., 2013; Shields, 2000; Wyllie, 1996, 1999).

Epileptic encephalopathy: Currently, epileptic encephalopathy is the most commonly used term. Epileptic encephalopathy has an ILAE-accepted definition (Berg et al., 2010), and encompasses the concept that epileptic activity contributes to cognitive impairment over and above the underlying cause. There are many examples of this phenomenon in the literature (described above in section 1.4.2), demonstrating that longer exposure to seizures and EEG abnormalities is associated with more severe developmental delay, that impairment can worsen over time, and that deficits may be reversible if the epileptic activity can be terminated early. The consequences of epileptic encephalopathy are most severe in infants and young children, as disruption of normal brain activity impairs not only existing brain networks but also the development of mature networks. The concept of epileptic encephalopathy is critical for clinicians to

understand. Recognition of epileptic encephalopathy in a patient has major implications for treatment; aggressive therapy is warranted not only for seizure control but also to minimise developmental impacts (rev in (Howell, Harvey, & Archer, 2016)).

In practice the term epileptic encephalopathy is used in two distinct but related and overlapping ways. The first considers epileptic encephalopathy as a concept (as described above); the phenomenon of epileptic encephalopathy may or may not be present in an individual patient (Engel & International League Against, 2001). The second use of the term is as a ‘classifier’ for epileptic syndromes which are considered to be severe and frequently have a component of epileptic encephalopathy. A number of established neonatal and infantile epilepsy syndromes, such as EIEE, EME, EIFMS and West syndrome are considered to be epileptic encephalopathies (Berg et al., 2010).

There are a number of potential issues relating to use of the term epileptic encephalopathy in clinical practice or research (rev in (Howell et al., 2016)). It can be difficult to establish whether some patients fit the definition of epileptic encephalopathy at any point in their condition. It is often hard to determine whether, or to what extent, the cognitive impairment is a result of the epileptic activity (versus the underlying cause or AEDs). The term epileptic encephalopathy does not have strict diagnostic criteria that can be easily applied early in the epilepsy to all patients given its main measure, cognitive impairment, may not be apparent until the child is older.

Catastrophic epilepsy: Catastrophic epilepsy is another term used for ‘severe’ epilepsies, initially used in North American studies of epilepsy surgery candidates (Wyllie, 1996). The most significant problem with this term is the lack of a single accepted definition; many different definitions are used in the literature, again variably at individual patient and syndrome levels (Oguni et al., 2013; Shields, 2000; Wyllie, 1996, 1999). At the individual patient level, definitions typically incorporate frequency and intractability of seizures +/- impact on development. At the syndrome level, catastrophic epilepsy has been used for any progressive epilepsy regardless of whether the epilepsy or the underlying condition is the primary cause for the decline. For example, conditions such as progressive myoclonus epilepsy and other neurodegenerative disorders would be considered catastrophic epilepsy, but would not

necessarily meet the definition of epileptic encephalopathy. A further issue with the use of catastrophic epilepsy is that the term implies a terrible prognosis to patients and families.

Refractory epilepsy: The definition of refractory epilepsy has also varied in the literature over time, but more recently a standard definition has been agreed upon (Kwan & Brodie, 2010). Most definitions of refractory epilepsy incorporate a failure of a two or more AEDs +/- a specific ongoing seizure frequency or failure to achieve a duration of seizure freedom (rev in (Berg & Kelly, 2006)). It is important to note that refractory epilepsy is not necessarily the same as ‘uncontrolled’ epilepsy, which reflects ongoing seizures when the criteria for ‘refractory’ epilepsy are not met (e.g. ongoing seizures in a patient not on AED treatment). It is also not necessarily the same as ‘persistent’ epilepsy, as some epilepsies that are refractory for a time will eventually remit.

A study comparing different published definitions (including a number used in paediatric cohorts) showed variable agreement between the definitions, but that all were associated with ultimate remission status rev in (Berg & Kelly, 2006). The authors suggested that it might be worthwhile having an ‘official’ definition for comparability, but also that different definitions might be required for different clinical settings and questions. The standard definition put forward by the ILAE Commission on Therapeutic Strategies defines refractory epilepsy as failure of adequate trials of two tolerated, appropriately chosen and used antiepileptic drug schedules to achieve sustained seizure freedom(Kwan & Brodie, 2010). The main issue with this term in ‘severe’ infantile epilepsies is that its use as a single criterion is not specific for poor epilepsy and developmental outcomes.

Comparison of definitions: Table 1.7 compares the epilepsy, EEG and developmental criteria used for each of the previously discussed definitions of ‘severe’ epilepsy.

Table 1.7 Comparison of definitions of the terms used in the literature for ‘severe’ epilepsies

Seizures EEG abnormalities Development

Epileptic Includes ‘epileptic activity’ which Includes frequent Change in/impact on encephalopathy incorporates seizures, but ‘epileptic activity’, development due to doesn’t make specific which incorporates the epileptic activity statements re epileptiform EEG frequency/intractability etc. abnormalities (rather just frequent ‘epileptic activity’ that impacts on development)

Catastrophic Most definitions include a - Most definitions epilepsy criteria re frequent and/or include a criterion intractable seizures that includes a change in/impact on development (but this doesn’t necessarily have to be due to the epilepsy)

Refractory Pharmacoresistant seizures - - epilepsy

The variable use of the aforementioned terms highlights the lack of a consensus definition for ‘severe’ epilepsies that can be easily applied to all patients early in the course of the condition.

A definition of ‘severe’ epilepsies is needed to highlight the group of infants with the potential to have frequent and drug resistant seizures, significant impacts on development, and a major burden on families and society. This patient group is different to the group of infants with FS and ‘benign’ epilepsies, who predominantly have favourable outcomes. Identifying infants with ‘severe’ epilepsies is important for guiding investigations and treatment, putting in place appropriate supports for the infant and their family, and further research into novel treatments to improve outcomes for these infants.

Given this, it is important to consider how ‘severe’ will be defined in this study, and whether a novel definition may be useful.

1.9.2 Factors that could be considered in a novel definition of ‘severe’ epilepsy of infancy An ideal definition of ‘severe’ epilepsy in infants would be predictive of the intractable epilepsy and/or poor developmental outcomes that characterise this group. It would have clear diagnostic criteria that could be identified at presentation or early in the course of the epilepsy, at the time at which decisions about investigation and treatment are being made. This section considers epilepsy and development-related factors that could be included in such a definition.

Development-related factors: Multiple studies show that prior development is a strong predictor of later development in infants with epilepsy. However, the use of criteria that include developmental status is problematic in a ‘severe epilepsy’ definition given:

 impairment may not be apparent at initial presentation, especially in young infants  some infants have definitively normal development at seizure onset  it is one of the outcomes we would like this term to predict, and a priori assumptions about developmental prognosis may bias the term  developmental delay plus seizures does not necessarily indicate a ‘severe’ epilepsy (e.g. some delayed children may have infrequent seizures only in the setting of a static encephalopathy) or one that contributes to cognitive outcome over and above the underlying cause  presence of a developmental plateau or regression at seizure onset is not reported as an independent predictor of developmental outcome (in studies of patients with infantile spasms, or groups of all infants with epilepsy).

Epilepsy factors: Epilepsy-related predictors of poor seizure and developmental outcome in childhood epilepsy are epileptic syndrome, age of seizure onset, seizure frequency, interictal epileptiform abnormalities, pharmacoresistance/refractory seizures and duration of epilepsy (including delays to treatment). Of these, the factors that could be useful in the infant population (specifically which could be applied to all patients early in the course of the epilepsy) are frequent seizures, IEDs and pharmacoresistance (Altunbasak, Incecik, Herguner, & Refik Burgut, 2007; Berg et al., 2014; Berg et al., 2004; Berg et al., 2012; Cavazzuti, Ferrari, & Lalla, 1984; Cormack et al., 2007; Datta

& Wirrell, 2000; Eisermann et al., 2003; H. Freitag & Tuxhorn, 2005; Kivity et al., 2004; Loddenkemper et al., 2007; Matsumoto et al., 1983; O'Callaghan et al., 2011; Vasconcellos et al., 2001; Vendrame et al., 2009; Zaroff et al., 2004). Further detail is provided in Table 1.8.

Table 1.8 Applicability of epilepsy prognostic factors to the infant population

Can be applied to all patients early in Measure Additional notes course of the epilepsy? Epileptic syndrome No Well-defined epileptic syndrome not present in ~40-50% Where well-defined syndrome present, syndrome is strongly predictive of outcome

Age of epilepsy Yes But, not helpful in this instance as the age of the study onset population (i.e. infancy) is already determined

Seizure frequency Yes *see below

Interictal Yes Presence of IEDs is correlated with poor seizure and epileptiform discharges (IEDs) developmental outcome in a number of studies of epilepsy in infants Conceptualised in definitions of epileptic encephalopathy as contributing to poor outcome

Pharmacoresistant/ Yes Defined as ongoing seizures despite adequate trials of refractory epilepsy at least 2 appropriate AEDs Highly sensitive for poor outcome (FSIQ <80 in 87% of pts with drug resistant epilepsy onset <1yo and ~80% in pts with onset <2yo, also, declining IQ seen in pts 0-3yo with pharmacoresistant epilepsy in the first 3 years of their epilepsy, but not in those with well-controlled seizures) Not specific for poor outcome (e.g. in case of Rx- responsive West syndrome) *Sometimes number of AEDs used is considered separately from treatment-response, but given the interaction between these two measures, they are considered together here

Duration of epilepsy No (yes if delay to Longer periods of epilepsy predictive of poorer (including delays to treatment) treatment initiation) outcomes, but can’t apply duration of epilepsy prospectively in most cases (delay to treatment predicts poorer outcome, but prompt treatment doesn’t mean normal outcome)

While there is a standard definition of pharmacoresistance, and IEDs could be categorised as present or absent, it is less clear how ‘high’ and ‘low’ seizure frequency would be best defined. As such, it is worth considering further the spread of seizure frequency in infantile epilepsies, and the relationship of seizure frequency to outcome in studies in at this age group.

A US study of infants aged 1-12 months presenting with afebrile seizures to a single US hospital between 1994-1998 identified greater than weekly seizures in 68%, weekly- monthly seizures in 0%, less than monthly seizures in 2.5% and 1-2 seizures only in 30%(Datta & Wirrell, 2000). A similar study in Turkey of all infants aged 1-24 months with epilepsy presenting over a 5 year period to a single hospital for whom follow-up data was available showed greater than weekly seizures at presentation in 76% and less than weekly seizures in 24% (Altunbasak et al., 2007). The studies that relate seizure frequency to developmental outcome are listed in Table 1.9. These studies suggest that the higher the seizure frequency, the higher the rates and severity of developmental impairments. While the categorisation of seizure frequency (and number of patients in each category) varied from study to study, most studies report high rates of poor developmental outcome in those with weekly or greater seizures. The data for less than weekly seizures is less clear. One large study suggests high rates of poor outcome for patients with greater than monthly seizures occurring over an 18 month period(Berg et al., 2012). However, it is important to note that this was done in patients with pharmacoresistant epilepsy, and it is not clear whether these data (particularly those relating to lower seizure frequencies such as greater than monthly) can be extrapolated to infants who do not meet pharmacoresistance criteria, or who have a shorter duration of epilepsy. The shortest duration of frequent seizures required to impact on developmental outcome is also not clear, although progressive developmental impacts are noted patients with infantile spasms as the duration of seizures increases, with mild impacts detectable even with short duration of seizures (Humphrey, Williams, Pinto, & Bolton, 2004; O'Callaghan et al., 2011).

Table 1.9 Studies looking at the relationship between seizure frequency and developmental outcome

Seizure frequency Study Patient group Outcomes categorisation

Vasconcellos, Patients evaluated for Daily, weekly, monthly IQ < 70 in 65% with Epilepsia 2001 epilepsy surgery with (NB analysed as daily daily seizures uncontrolled epilepsy in the (n=20) vs IQ <70 in 22% with context of a focal lesion weekly/monthly (n=9) in weekly/monthly limited to one lobe (a subset patients <24 months old seizures (NB small of which had epilepsy onset as onset) number of patients and at <24 months old), IQ breakdown of numbers testing done at >5 years old in weekly vs monthly in 96% groups not provided)

Oguni, Brain Dev Patients with greater than >10 seizures per month 62% of patients with 2013 10 seizures per month (NB onset at <6 years had this was an inclusion criteria poor outcome (data for for a study on catastrophic infant onset group only epilepsy, authors note was not provided) somewhat arbitrary choice of this seizure frequency)

Berg AT Patients with > monthly seizures over High risk of Neurology 2012 pharmacoresistant epilepsy an 18 month period (part developmental of their criteria for impairment, decline in pharmacoresistance) IQ over time in younger patients

Humphrey A, Patients with tuberous Weekly, daily, more than All groups had Epilepsy sclerosis age 11-36 months daily cognitive impairment, Research 2008 but seizure frequency and Eur Child correlated with Adol Psych 2004 severity

A precisely-defined consensus definition for ‘severe’ epilepsies in infancy that can be applied early in the course of the epilepsy is desirable, both at the individual patient level to inform prognosis and guide management, and at a broader level to allow comparability between studies in this population. Three epilepsy related factors, frequent seizures, IEDs and pharmacoresistance, are predictive of outcome and can be determined early in the course of the epilepsy. These factors would potentially be useful in a definition of ‘severe’ epilepsy of infancy, although none can individually predict poor outcome in all patients. As such, any novel definition should consider

incorporating more than one of these factors. The definition of ‘severe’ epilepsy of infancy used in this study is further discussed in Chapter 3.

1.10 Conclusions

Infant epilepsies are a highly heterogeneous group of disorders, with variable phenotypes, and outcomes, due to a large number of acquired and genetic aetiologies.

There appears to be a dichotomy between infants with ‘benign’ and ‘severe’ epilepsies, with relatively few ‘intermediate severity’ infant epilepsies. The well-described epileptic syndromes of infancy have either almost universally favourable (e.g. benign neonatal, neonatal/infantile and infantile familial epilepsies) or almost universally poor (e.g. EIEE, EIMFS) outcomes.

Infants with ‘severe’ epilepsy have frequent seizures, an epileptiform EEG and typically, significant developmental impairments that are likely to be, at least in part, due to the damaging effects of seizures on the developing brain. For many, current treatments are not effective. There is an urgent need for novel treatments in this group of infants.

There are many gaps in knowledge of the incidence, clinical features, aetiologies and diagnostic investigation of ‘severe’ epilepsies, as a group and within particular epileptic syndromes, that limit our ability to improve outcomes in affected infants.

The cause remains unknown in many infants despite extensive imaging, chromosomal and metabolic testing; these infants have a presumed genetic basis. This information is needed in order to inform prognosis and guide treatment for patients, and allow accurate reproductive counselling for families. Understanding the causes is a major step towards development of novel and targeted treatments.

While the incidence of some infant epileptic syndromes such as West syndrome and Dravet syndromes has been studied, that of the whole group of ‘severe’ epilepsies has not. In particular, there is no genetic epidemiology to make sense of which of the many

reported genes are more important at a population level. This information is important to guide priorities for research in order to provide benefit to as many infants as possible.

The clinical features of many genetic causes of ‘severe’ epilepsy are incompletely characterized, and many do not fit strict criteria of prototypic epileptic syndromes. A better understanding of the phenotypes will increase early diagnosis, improve ability to determine the clinical significance of variants identified in epilepsy genes in affected infants, understand prognosis and identify strategies for screening for likely comorbidities. In some cases, clinical observation of phenotypes has guided further research into mechanisms of, and treatments for, these conditions.

With increasing recognition of genetic causes, genetic testing is indicated in clinical practice, although access to such testing remains limited. While the yield of next- generation genetic testing such as WES is reported in referred patient groups, no population-based studies have been performed, and the cost-effectiveness of WES compared with current standard diagnostic testing in these infants has not been investigated. A knowledge of both the causes and the incidence of ‘severe’ epilepsies is required to inform implementation of optimal diagnostic pathways in clinical practice and optimize early aetiologic diagnosis.

This study of ‘severe’ epilepsies in infants addresses some of these unanswered questions. The study aims, design and definitions are discussed in the following chapters.

Chapter 2: Introduction, aims and hypotheses

2.1 Introduction

In the previous chapter, I reviewed the literature on infant epilepsies, noting the incidence of these conditions and the composition of this patient group, and highlighting groups of infants with favourable and poor outcomes. I discussed the well- established epileptic syndromes and described their associations with particular aetiologies, comorbidities and outcomes, emphasising that epilepsy in many infants does not fit an established syndrome. I reviewed the increasing discovery of genetic aetiologies in infants with unknown cause, and the recent use of NGS for aetiologic diagnosis.

I finished by focussing more specifically on definitions of ‘severe’ epilepsies of infancy, noting that there is not currently a widely-used, standard definition of ‘severe’ epilepsy that can be applied to all infants early in the course of their epilepsy. I studied factors which predict poor outcomes that could be utilised in a novel definition; in the Methods chapter of this thesis, I describe how these data were used to devise such a definition for this study.

There is an imperative to better understand the ‘severe’ epilepsies, given outcomes are poor and treatment is often ineffective. Until now, no study has systematically studied the whole group of infants with ‘severe’ epilepsies. A more complete understanding of the aetiologies of these conditions is needed to optimise early diagnosis and develop novel therapies. The genetic epidemiology must be studied to make sense of which of the many causes, particularly genetic causes, are most important at a population level, in order to guide research priorities and optimise yield and cost of diagnostic pathways. The increasing availability of WES in clinical practice makes this study timely.

This thesis reports a population-based study of the epidemiology, causes, clinical features and diagnostic investigation of SEI in infants born during 2011-2013 in Victoria, Australia. I performed a detailed study of the epileptology and outcomes, and

systematic review of brain imaging. WES was undertaken in infants whose aetiology was unknown.

2.2 Aims

The specific aims of the study were to:

 Determine the incidence of severe epilepsies of infancy (SEI) and specific epileptic syndromes  Identify the aetiologies of SEI at a population level  Study the clinical features of infants with SEI until age two years  Determine the highest yield and most cost-effective protocol for making an aetiologic diagnosis by determining if and when WES should be included in the diagnostic pathway

2.3 Hypotheses

The hypotheses tested in this study were:

 SEI are not uncommon disorders in infancy, with significant health burden for families and society  West syndrome is the most common epileptic syndrome, and focal epilepsies predominate in the remaining infants  Genetic disorders of brain structure and function are more common causes of SEI than acquired factors  WES performed early in the investigation of SEI is cost-effective in reaching an aetiologic diagnosis

Chapter 3: Methods

3.1 Introduction

This study was a population-based, epidemiologic, phenotypic and genetic study of severe epilepsies of infancy (SEI) in infants born in the state of Victoria, Australia, during the three years 2011-2013, with onset of SEI between January 1, 2011 and June 30, 2015. The study was conducted during 2013-2016. SEI is defined in the Inclusion Criteria section below.

This study aimed to determine the incidence, and study the clinical features and aetiologies of SEI. Aetiologies were pursued in infants with SEI of unknown aetiology following clinical diagnostic testing, with a focus on genetic causes and brain malformations. The cost and diagnostic yield of gene panel testing was compared with current standard investigations, namely brain imaging, chromosomal and metabolic testing.

Ascertainment of infants with potential SEI occurred via a number of sources, namely Victorian EEG laboratories, databases from tertiary NICUs and referrals from paediatric neurologists. Ascertainment and confirmation of SEI informed the epidemiologic aspect of the study.

Assessment of infants with SEI was undertaken to clarify the electroclinical phenotype and determine aetiology. Assessment involved detailed review of medical records, EEGs, brain imaging and diagnostic investigations on all infants with SEI. Face-to-face assessments and genetic testing were undertaken on infants with SEI of unknown aetiology. The study design diagram (Figure 3.1) provides further description.

Figure 3.1 Study design diagram

3.2 Study population

The study population consists of all infants born in Victoria during 2011-2013 with SEI.

Victoria is the second most populated state of Australia. In 2011, the population was 5,582,670 and live birth rate 71,444. 6% of the population was under 5 years old. Victoria is highly multicultural; in 2011 only 69% of Victorian residents were born in Australia and 23% spoke a language other than English at home. Victorian residents reported ancestry (within three generations) in over 200 countries; the top 5 nominated ancestries, English, Australian, Irish, Scottish and Italian, made up 84% of the population. 75% of the population lived in the Greater Melbourne area, the remainder in regional or rural areas. 53,683 more people migrated into Victoria (from interstate and overseas) than migrated out, including 3,755 children under 5 years old. The average maternal age at birth was 30.7 years old. Most births occurred in hospitals, with the home birth rate just 0.8%. The rate of lower uterine segment caesarean section was 33%. Infant mortality was low, at a rate of 3.5 deaths/1000 live births. The average weekly wage was $966.80 (www.abs.gov.au).

Access to both publicly and privately-funded health care is good. All Australian citizens, and citizens of countries with reciprocal medical care arrangements, have access to ‘Medicare’, a government funded universal health care scheme that enables access to free treatment in public hospitals and subsidized treatment by medical practitioners with a Medicare provider number, including general practitioners, paediatricians and paediatric neurologists (www.mbsonline.gov.au).

Primary health care of infants in the community is provided by general practitioners (GP) and maternal and child health nurses (MCHN). MCHN practice at government- funded local health centres. Families are put in touch with a local MCHN at the time of their child’s birth, and most attend for growth checks and basic developmental screens at intervals in infancy. Concerns identified at these visits are typically referred on to a GP. GPs typically work at privately-owned (but partly government-funded) practices in the community. Most families have a GP (either an individual doctor or a particular practice) that they attend when required, but not all infants see a GP. Potential reasons for a GP appointment include the 6-week infant check (not all attend), immunisations

(most go to local council vaccination sessions) or for illness and other health and developmental concerns. Infants born in rural and some regional areas will often have a neonatal health check with a GP. GPs may refer a child to a paediatrician or a neurologist. While infants born in tertiary and some secondary hospitals typically see a paediatric doctor in the neonatal period (paediatrician in private hospitals, usually a paediatric registrar in public hospitals), only some will see a paediatrician subsequent to discharge from hospital, typically where there is a particular health concern and the GP and/or family seek specialist paediatric advice. Families can see a paediatrician either through a public hospital outpatient clinic, or at private practices in the community. Paediatricians are able to refer to a neurologist. Only a small proportion of infants will see a neurologist, however most infants with a neurologic disorder will. While most neurology outpatient services are provided through public hospitals, some paediatric neurologists do outpatient clinics in private practices. Inpatient general paediatric services are available at both public and private hospitals. However, apart from perinatal services (approximately 28% of infants are born in private hospitals), the vast majority of paediatric (and all neurologic) inpatient care occurs in public hospitals.

Primary health care is available in all suburbs of Melbourne, all regional centres, and many rural towns. Paediatricians are available across Melbourne, and in most regional centres. Most neurologists practise only in Melbourne, although outreach clinics are available in some regional centres. Public hospitals with paediatric services are available in Melbourne and in larger regional centres. Inpatient neurology units and tertiary neonatal care are available only in Melbourne. Funding to subsidise travel for medical care is available via the Victorian Patient Transport Assistance Scheme to all residents of rural towns required to travel more than 100km one way to access specialist medical treatment.

Brain imaging is primarily requested by neurologists and paediatricians. GP access to MRI brain imaging is limited, although ‘unexplained seizures’ is a Medicare-funded indication for GP-referred MRI (www.mbsonline.gov.au). Due to the requirement for anaesthesia for most infant MRI scans, most scans are performed in tertiary public hospitals in Melbourne with access to specialist paediatric anaesthetic services. Any

doctor can refer an infant for an EEG, and these are performed at nine sites in Melbourne and regional Victoria.

Access to antiepileptic therapies in Victoria is good. Most anticonvulsant medications are available. Costs for a large list of medications are subsidized by the government- funded Pharmaceutical Benefits Scheme (www.pbs.gov.au), which is available to all Australian residents with a Medicare card. The ketogenic diet is offered at three centres in Melbourne. RCH has a large epilepsy surgery program that has performed over 500 epilepsy surgeries and is the major site for paediatric epilepsy surgery in Australia. Approximately 50 surgeries are performed per year currently, including on infants over 5kg, and infants and children with both complex (e.g. TS, multifocal dysplasias) and subtle (e.g. bottom of the sulcus dysplasias) brain malformations.

3.3 Inclusion criteria

Much thought was given to the definition of SEI for this study, to ensure recruitment of the expected patient population. Given the absence of an accepted definition of ‘severe’ epilepsy in infancy, the difficulties of applying the most widely used term ‘infantile epileptic encephalopathy’, and the absence of a single factor that informs ‘severity’ of epilepsy or developmental outcomes, a multifactorial definition of SEI was used, based on the epilepsy-related factors discussed in Section 1.9.2 (Methods chapter).

Three criteria considered appropriate for a definition of SEI, which relate to the epilepsy specifically, and not development, and which could be applied to all patients early in their epilepsy were high seizure frequency, pharmacoresistance and prominent epileptiform abnormality on EEG.

Pharmacoresistant epilepsy was defined as ongoing seizures despite efficacy failure of two appropriate AEDs used in appropriate doses(Kwan & Brodie, 2010). Frequent seizures was defined as at least weekly seizures for a month OR daily seizures for a week. Interictal epileptiform abnormality was defined as presence of interictal or ictal epileptiform discharges on EEG, regardless of type or abundance given difficulties with quantifying EEG.

It was important to consider whether all three criteria were necessary to the definition of ‘severe’ epilepsy in infants. Such a definition would ideally result in the inclusion of patients with the ‘severe’ epileptic syndromes such as EIEE and West Syndrome and would exclude those with ‘benign’ (self-limited, pharmacoresponsive) epileptic syndromes. Table 3.1 considers how these criteria apply to patients with well-described infantile epilepsy syndromes.

Table 3.1 Potential criteria for a definition of ‘severe’ epilepsy in patients with well- described infantile epileptic syndromes

Pharmacoresistant Interictal EEG Frequent seizures epilepsy abnormality

WANT TO INCLUDE:

EIEE + + +

EME + + +

EIMFS + + +

+ (depending on +/- + West Syndrome duration ) +/- (but may not get a -/+ (may develop -/+ (may develop Dravet syndrome diagnosis of epilepsy during second year of during second year until second year of life) life) of life) ‘Structural’/ ‘symptomatic’ focal +/- +/- + epilepsy* WANT TO EXCLUDE:

BFNE/BFNIE/BFIE - -/+ -/+

BMEI - + -/+

Based on the above, it was decided that all three criteria would be used to define SEI for this study. The use of all three criteria would be most effective in excluding patients with benign epileptic syndromes and presentations, and would include most patients whose epileptic syndromes would generally be considered severe. Importantly, a requirement that multiple inclusion criteria be met limits the possibility of ‘regression to the mean’ ‘diluting’ the desired SEI population. Studies of severe conditions, particularly conditions such as epilepsy in which fluctuations of severity occur, have the potential to be complicated by this phenomenon, which notes that some initial extreme

or severe measurements tend to move closer to average on repeat testing. Thus, undesired inclusion of infants whose epilepsies have a brief period of being ‘severe’ but subsequently settle, such as those with ‘benign’ focal epileptic syndromes, is much less likely to occur than if inclusion were based on fewer criteria.

Patients that might not be covered by this definition include those with treatment- responsive infantile spasms. It was decided therefore that infantile spasms would be an additional ‘automatic’ inclusion criterion given poor outcomes in most such patients, even those with short duration of seizures or treatment response.

Additionally, many patients with Dravet syndrome or ‘structural’ focal epilepsy would not meet these criteria. While most of these patients have epilepsy that is ultimately ‘severe’, it may not be so in infancy (e.g. in Dravet syndrome seizures in infancy may be months apart and the EEG normal, and those with structural focal epilepsies may have infrequent infantile seizures and become ‘refractory’ after infancy). It was therefore decided that these would not be additional ‘automatic’ inclusion criteria.

Finally, these criteria would not capture infants with recurrent bouts of status epilepticus unless extremely frequent or with other seizures in between bouts of status. Examples include some infants with Dravet syndrome and Sturge Weber syndrome. Recurrent status epilepticus was not considered to automatically infer an SEI given a) its association with poor seizure and developmental outcomes is less strong than the aforementioned factors and b) infants whose episodes of status epilepticus occur only while febrile will not meet a definition of epilepsy. Additionally, as not all infants with status epilepticus will have an EEG (e.g. those with febrile status epilepticus), the identification of infants with recurrent status epilepticus would be expected to be incomplete, and impact on the strength of the epidemiologic data. As such, recurrent status epilepticus was not an ‘automatic’ inclusion criterion.

Examining the performance of the chosen definition for SEI in comparison to alternative definitions, such as those that that include Dravet syndrome +/- recurrent status epilepticus, is of interest, but beyond the scope of this study.

Finally, it should be explicitly stated that the definition of epilepsy used in this study is two or more unprovoked seizures occurring more than 24 hours apart. As is standard, infants with only FS or acute symptomatic seizures are not considered to have epilepsy (Fisher et al., 2014; Fisher et al., 2005).

Thus, in summary, the inclusion criteria used in this study were:

 Born in Victoria between January 1, 2011 and December 31, 2013  Epilepsy with onset of seizures before age 18 months old  and EITHER:

o 1) Frequent seizures (≥ daily for one week or ≥ weekly for one month) 2) ongoing seizures despite trials of two appropriate AEMs, and 3) an epileptiform abnormality on EEG

o Infantile spasms

3.4 Ascertainment

Potential infants with SEI were ascertained retrospectively and prospectively from multiple sources, as no single method of screening would capture all potentially eligible infants in the state. Medical records of infants for whom source information suggested a possible SEI were reviewed to determine whether clinical and demographic inclusion criteria were met. Where this could not be determined from the medical records, clinicians were contacted and families interviewed to clarify this. Ascertainment and confirmation of SEI informed the data on SEI epidemiology.

3.4.1 Ascertainment Sources Paediatric EEG laboratories. Victorian EEG laboratories that perform EEGs on infants are RCH, MMC, Austin Hospital, regional hospitals in Geelong, Ballarat, Bendigo and Shepparton, and in the regional towns of Warrugal and Frankston. The two largest sites, RCH and MMC, perform approximately 1700 and 900-1000 paediatric EEGs per year respectively. EEGs are performed using the 10-20 electrode placement

system for most infants over one month old, and neonatal electrode placement was used for most neonates. Routine EEG recordings last 20-60 minutes, and prolonged studies performed to record seizures last 60 minutes to five days. EEGs are digitally recorded using Profusion software (Compumedics). Concurrent video recording is performed for all studies at RCH, MMC and the Austin Hospitals. After EEGs are reported clinically, video recordings during interictal periods are discarded, but video of clinical episodes are kept and available for review. Concurrent video recording is not undertaken at some regional centres. Sedation is rarely used to obtain an EEG recording.

Victorian paediatric neurologists. Victoria has 16 paediatric neurologists, who manage children with a variety of neurologic problems, including most children with ‘non-benign’ epilepsies. All paediatric neurologists work primarily in Melbourne, predominantly in public hospitals (RCH 12, MMC 4, Austin 1), with some also in private practice (8). Three paediatric neurologists perform regional outreach clinics in Geelong, Ballarat and Warrugal. Six paediatric neurologists are also epileptologists, having undertaken subspecialty epilepsy training at major paediatric epilepsy centres. The study was widely advertised to neurologists, and frequent reminders to refer potentially eligible patients were sent.

Neonatal intensive care units. Victoria has NICUs at RCH, MMC, RWH and MHW, all located in Melbourne. NICUs manage all infants less than 32/40 gestation, all intubated infants, all neonates requiring surgery and those with complex medical conditions. There are a number of secondary neonatal units in Melbourne, and in larger regional centres. Discussion with neonatologists, including those working with the Neonatal Emergency Transport Service, indicated it was rare for a neonate with seizures to be managed outside of a tertiary centre.

Figure 3.2 Study screening sites

Figure shows A) Victorian metropolitan screening sites and B) Victorian regional screening sites. Red dots represent EEG laboratories, green dots NICUs and yellow dots paediatric neurologists.

3.4.2 Search strategy EEG reports. EEG reports from all EEGs performed in the aforementioned Victorian EEG laboratories on children under two years old during 2011-2015 were reviewed. All EEGs were reported by a neurologist, all but one of whom is a paediatric neurologist. Six of ten neurologists are also epileptologists. The information available on the EEG report is a brief clinical summary, detailed information of the electroclinical features seen on EEG including whether an episode occurred during the EEG, and a conclusion which interprets the electroclinical findings. Infants were designated one of nine categories based on the clinical detail and EEG findings documented in the report: not seizures, unlikely seizures, possible seizure but not SEI, seizures but not SEI, FS, possible seizures in a neonate, possible seizures and SEI, possible SEI, and SEI. Infants in the first five categories were deemed to have non-epileptic or benign epileptic conditions, and were not further studied. Hospital medical records of infants in the latter four categories were reviewed to determine which infants met or might meet clinical and demographic inclusion criteria. Infants in the latter four categories included all infants with an epileptiform EEG, and any infant in whom the clinical features indicated developmental regression or frequent episodes that were not obviously benign (examples of benign conditions included sleep myoclonus and shuddering).

Victorian paediatric neurologists. Victorian paediatric neurologists were asked to notify me of potential participants at the time of diagnosis or review. Where Victoria neurologists notified the primary investigator of potential participants, clinical details and EEGs were discussed and reviewed with the neurologist, and hospital medical records reviewed, to determine infants with possible or definite SEI.

NICUs. All neonates with seizures at the four Victorian tertiary neonatal units were identified through searches of departmental databases. Three of the four Victorian NICUs have a comprehensive database of all admitted infants which codes information including admission and discharge diagnoses, and associated medical problems. All three contained a ‘seizures’ field (coded as yes/no) and were therefore searched for ‘seizures – yes’. MMC does not have such a database, but has a repository of all discharge summaries which can be searched for keywords (in this case ‘seizure’). Medical records of all neonates with seizures were reviewed, and infants designated one of seven categories: not seizures, unlikely seizures, possible seizures but not SEI, seizures but not SEI, possible seizures and SEI, possible SEI, and SEI. Patients in the first four categories were deemed to have non-epileptic or benign epileptic conditions, and not further studied. Infants in the latter three categories were deemed to have possible or definite SEI, and medical records further searched to determine if demographic inclusion criteria were satisfied.

3.4.3 Confirmation of SEI diagnosis The diagnosis of SEI and satisfaction of demographic inclusion criteria was confirmed on review of medical records as noted above. Where uncertainty remained following medical record review as to whether an infant met clinical or demographic inclusion criteria, EEG recordings were reviewed and further information was obtained through the treating doctor. Where uncertainty still remained, families were contacted to clarify inclusion, with further information obtained by phone interview or face-to-face interview.

3.5 Assessment and analysis of individual patient data

Historical, examination and investigational data were collected on all infants with definite SEI to determine the aetiology and characterize the electroclinical phenotype.

Review of medical records, EEG recordings, brain MRI and other diagnostic investigations was performed for all infants.

Additionally, infants with SEI of unknown aetiology or uncertain electroclinical phenotype, and those who had not been assessed by a Victorian paediatric neurologist were invited to undergo a face-to-face clinical assessment to provide additional clinical data and clarify historical records. Face-to-face assessment was not performed for infants with a definite aetiology and electroclinical phenotype, such as infantile spasms secondary to well documented perinatal hypoxic ischaemic injury, TS and trisomy 21.

Infants with SEI of unknown aetiology underwent genetic testing.

Table 3.2 Data used to determine aetiology and electroclinical phenotype

Electroclinical Aetiology phenotype History and examination: medical record + + review

History and examination: face-to-face +/- +/- assessment

EEGs + N/A

Brain MRI N/A +

Other diagnostic investigations N/A +

Research genetic testing N/A +/-

+ = performed in all infants, +/- = performed in some infants, N/A = not applicable

3.5.1 Medical record review Medical records from the site of identification, including inpatient admission and outpatient clinic notes, were reviewed in detail. Permission was sought to obtain any records held at other sites from

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Table 3.3 Clinical and demographic data obtained from the medical record

Section Data obtained Detail of categorisation

Demographics Gender Male, female, indeterminate Family history Epilepsy (in first or second degree relatives), individuals with similar severe epilepsies Consanguinity Yes (nature) or no Age at assessment Age in months or years Duration of follow-up Age in months or years Aetiologies Aetiologic diagnosis Specific diagnosis, with genetic confirmation where relevant Aetiologic classification Modified version of 2010 international League Against Epilepsy diagnostic classification scheme using the following aetiologic groups: acquired conditions, brain malformations (as subdivisions of ‘structural’ group), metabolic disorders, chromosomal abnormalities and single gene disorders (the latter two being subdivisions of ‘genetic’ group)

Timing of diagnosis (pre- Pre-seizure onset, post-seizure onset or nil seizure onset, post-seizure diagnosis made onset) Method of diagnosis History/examination, standard clinical investigation (and which investigation), research genetic or imaging investigation (and which) Epilepsy Age of seizure onset Months (days if first week of life, weeks if first month of life) Seizure types at presentation See ‘review of seizures and EEG analysis’ section Seizure types at evolution See ‘review of seizures and EEG analysis’ section Epileptic syndrome at See ‘review of seizures and EEG analysis’ presentation section Epileptic syndrome at See ‘review of seizures and EEG analysis’ evolution section Antiepileptic treatment AEDs (number used, most beneficial drug), (number of AEDs, surgery, surgery (whether performed, type, outcome), ketogenic diet) ketogenic diet (whether undertaken, effect) Seizure outcome at 2 years Ongoing seizures or seizure free (for >3 old months) – on/off AEDs Development Pre-seizure onset Normal or delayed Plateau/regression at or after Yes or no epilepsy onset? Developmental outcome at 2 Normal or delayed years old Delay type (global or specific domain) Delay severity (severe-profound if skill level equivalent to <9 months old (i.e. DQ <35),

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Section Data obtained Detail of categorisation

mild-moderate if skill level equivalent to 9-21 months old) Visually attentive? Best expressive language skill Best gross motor skill Comorbidities Neurologic Head size, tone, hemiparesis, movement and disorder, other complications (normal or abnormal, type/distribution) Non-neurologic Neurocutaneous abnormality, macro/macrosomia, major organ abnormalities (yes or no, type) Long-term supplemental Yes or no feeding Survival At 2 years Alive or deceased At last follow up (age) Alive or deceased Detail of death Age at death Reason for death (clinical detail corroborated by post-mortem study where available) Diagnostic Brain MRIs Number performed investigations Findings (see ‘Brain MRI analysis’ section below) Diagnosis (where made) on routine clinical testing or research imaging review? Other routine diagnostic Tiers performed (and number of times each investigations performed performed, see Table 3.5 below for details of tiers) Significant findings Research genetic testing Which testing performed (single gene performed testing, molecular inversion probes gene panel, whole exome sequencing gene panel, other, nil) Significant findings

3.5.2 Review of seizures and EEG studies All available EEG recordings and seizure videos (both EEG and home videos) were reviewed, initially by me, and then jointly with my primary supervisor, Dr Simon Harvey, to reach a consensus on findings. Thus, EEGs were reviewed by two epileptologists.

Interictal background and epileptiform features, and ictal electrical and clinical features were considered in conjunction with clinical history to determine seizure types and epileptic syndrome at presentation (presentation defined as the time of presentation to medical care at which seizures were suspected or diagnosed), and if further features

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such as new seizure types or EEG features emerged. Seizure semiology was described. Seizure types were classified according to the 1981, 2010 and 2016 ILAE classifications (Berg et al., 2010; ILAE, 1981) (http://www.ilae.org/visitors/centre/Class-Seizure.cfm. Epileptic syndromes were defined as listed in Table 3.4, using ‘prototypic’ epileptic syndromes and ‘variant’ syndromes to classify epilepsies not consistent with a ‘prototypic’ syndrome according to the syndrome it most closely approximated.

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Table 3.4 Definitions of epileptic syndromes used in this study

Syndrome Clinical seizure type Interictal EEG Ictal EEG (where Age Other recorded) onset EIEE Tonic Burst-suppression Generalised <3m

EIEE-like Tonic Burst-suppression Focal <3m

EIEE-plus Tonic, other Burst-suppression Generalised, other <3m EME Myoclonic Burst-suppression Generalised <3m

EME-like Myoclonic Other (not burst- Generalised <3m Not meeting definition of myoclonic suppression) encephalopathy in a non-progressive disorder

EME-plus Myoclonic, other Burst-suppression Generalised, other <3m EIMFS Focal (multiple locations of Typically multifocal (but Focal (multiple locations of <6m Seizures ‘migrating’ between hemispheres and seizure onset, in both can be other) seizure onset, in both occurring frequently enough to constitute status hemispheres independently*) hemispheres independently*) epilepticus

104 EIMFS-like Focal (multiple types of Typically multifocal (but Focal, with seizures <6m Seizures may not be frequent enough to

seizures with different can be other) migrating between constitute status epilepticus semiologies reported without hemispheres but definite left and right independent left and right lateralisation of seizure onset) seizure onsets not demonstrated

EIMFS-plus (multi)Focal, other Typically multifocal (but Focal (multiple locations of <6m can be other) seizure onset, in both hemispheres independently), other

West syndrome Epileptic spasms Hypsarrhythmia/ modified Spasm complexes/ <18m With or without developmental hypsarrhythmia decrements plateau/regression

West Epileptic spasms Other (not Spasm complexes/ Any syndrome -like hypsarrhythmia) decrements West Epileptic spasms, other Hypsarrhythmia/ modified Spasm complexes/ <18m syndrome -plus hypsarrhythmia decrements, other

Syndrome Clinical seizure type Interictal EEG Ictal EEG (where Age Other recorded) onset West syndrome Epileptic spasms, other Other (not Spasm complexes/ Any -like-plus hypsarrhythmia) decrements, other

Dravet Febrile and/or afebrile Normal or generalised Generalised spike-wave, <15m Normal development before seizure onset syndrome generalized and/or unilateral spike-wave focal Resistant to antiepileptic treatment clonic or tonic-clonic seizures, with later seizures including myoclonus, atypical absences and focal seizures

Dravet Febrile and/or afebrile Normal or generalised Generalised spike-wave, <15m May have abnormal development before syndrome -like generalized and/or unilateral spike-wave focal seizure onset clonic or tonic-clonic seizures, +/- other

Myoclonic Myoclonic (episodes of Not burst-suppression Variable (mainly spike-wave) Any Absence of a progressive neurologic disorder encephalopathy myoclonic status), +/- other in a non-

105 progressive disorder

MAE Myoclonic-atonic, myoclonic or Generalised spike-wave Generalised spike-wave >7m Normal development before seizure onset atonic +/-other Normal MRI

MAE-like Myoclonic-atonic, myoclonic or Generalised spike-wave Generalised spike-wave >7 May have abnormal development before atonic +/- other months seizure onset May have abnormal MRI

LGS Multiple seizure types including Generalised slow spike- >1 year generalised tonic wave +/- paroxysmal fast activity

LGS-like Generalised +/- focal seizure Generalised or multifocal Any types

BMEI-like Myoclonic Normal Generalised spike-wave >3m Resistant to antiepileptic treatment

CAE-like Absences, other Generalised spike-wave Generalised spike-wave <2 years

Focal (uni) Focal (single location of seizure Normal or unifocal Focal (single location of Any onset) seizure onset)

Syndrome Clinical seizure type Interictal EEG Ictal EEG (where Age Other recorded) onset Focal (other) Focal (not definitely >1 location Multifocal or unifocal (must Focal Any Not meeting definition of unifocal, multifocal, of seizure onset) be multifocal if only a EIMFS or EIEE single reported seizure semiology) Includes unifocal seizures with multifocal EEG, and multiple seizure semiologies which are focal seizures and potentially referrable to >1 onset location (but not confirmed to be)

Focal (multi) Focal (multiple locations of Unifocal or multifocal Focal (multiple locations of Any Multiple locations of seizure onset determined seizure onset) seizure onset) EITHER through ictal recordings or through semiology localising seizure to different locations (i.e. each hemisphere – e.g. L and R clonic jerking, mirror image seizures) Not meeting definition of EIMFS or EIEE

GGE-other Generalised seizure types Generalised spike-wave Any Not meeting criteria for one of the other listed GGE syndromes (Dravet syndrome, Dravet

106 syndrome -like, BMEI-like, MAE, MAE-like, CAE- like)

* Independent seizure onset in both hemispheres confirmed (through ictal recordings or through semiology localising seizure to different locations (i.e. each hemisphere – e.g. L and R clonic jerking, mirror image seizures)

Shading: grey rows = prototypic ILAE syndromes, white rows = ‘variants’ of ILAE syndromes, light grey rows = other epilepsies. Bolded text in the variant syndrome rows notes the features that may be different to those of the related prototypic syndrome.

3.5.3 Brain MRI analysis MRI brain scans on infants in Victoria are performed primarily at RCH and MMC. A small number of scans are performed at the Austin and Geelong Hospitals, and at other suburban hospitals (Sunshine Hospital, Northern Hospital).

The RCH medical imaging department has two clinical MRI scanners 1.5Tesla (T) Magnetom Avanto, located in the medical imaging department), and 3T Siemens with intraoperative capability in the operating theatre complex) and one research MRI scanner (3T Magnetom Trio in the medical imaging department). MMC has 3 MRI machines (two 1.5T and one 3T, Siemens).

General anaesthesia was used for MRI on most infants to obtain images not affected by movement artefact. Some infants under 3 months old had imaging obtained while lying within an inflatable ‘beanbag’ that comfortably conforms to their body, restricting movement and typically facilitating sleep.

MMC and RCH employ specialist paediatric radiologists. RCH has two specialist paediatric neuroradiologists, including one with extensive experience in paediatric epilepsy and brain malformations.

MRIs were performed at 1.5 or 3T. Typical sequences performed included T2-weighted axial and coronal slices, T1-weighted volumetric acquisition reformatted in axial, coronal and sagittal views, and diffusion-weighted imaging, and susceptibility weighted imaging sequences. Fluid-attenuated inversion recovery sequences were performed in older infants, using either volumetric or orthogonal slices. Magnetic resonance spectroscopy was performed in some infants.

All brain MRIs in all infants were reviewed, initially by me and then in a joint review with a neuroradiologist, A/Prof Simone Mandelstam. Studies were reviewed systematically for abnormalities in the cortex, white matter, corpus callosum, basal ganglia, thalami, cerebellum and brainstem. Diagnostic and non-specific abnormalities were recorded. Clinical detail was used to inform MRI review where relevant (e.g. more detailed review of a particular cortical region if FCD suspected clinically).

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3.5.4 Other diagnostic investigations The results of all other diagnostic investigations were reviewed, including chromosomal testing, biochemical and metabolic studies of blood, urine and cerebrospinal fluid (CSF), and biopsies of skin, muscle and liver.

In Victoria, chromosomal testing including chromosomal microarray (CMA), banded karyotype and fluorescence in-situ hybridization are performed at the Victorian Clinical Genetics Service (VCGS). VGGS also performs sequencing for 23 common mitochondrial DNA mutations and 3 common (in Caucasian population) POLG mutations.

Much of the metabolic testing is performed at VCGS. This includes testing of urine for organic acids, amino acids, piperideine-6-carboxylic acid, S-sulphocysteine, guanidinoacetic acid and purines and pyrimidines, testing of blood for full blood examination (FBE), renal function (UEC), liver function (LFTs), glucose, calcium, magnesium, phosphate (Ca/PO/Mg), biotinidase, lactate, ammonia, amino acids, acylcarnitines, vitamin B12, copper, caeruolplasmin, uric acid and transferrin isoforms, and testing of CSF for cell count, protein, glucose, lactate, pyruvate and amino acids. Testing of very long chain fatty acids and lysosomal enzymes is performed at the National Referral Laboratory for Lysosomal, Peroxisomal and Other Related Disorders in Adelaide, South Australia. CSF neurotransmitters are measured at the Neurochemistry Laboratory of the Children’s Hospital at Westmead, Sydney. Electron microscopy of skin biopsies and histology of liver biopsies are performed at the Anatomical Pathology laboratory of the RCH. Muscle histopathology is performed at the Victorian Neuromuscular Laboratory at the Alfred Hospital. Respiratory chain enzyme analysis on muscle and liver tissue is performed at the VCGS.

3.5.5 Research clinical assessment Face-to-face clinical assessment was performed in infants with SEI of uncertain aetiology, where further detail was required to clarify the electroclinical phenotype, and in those who had not been assessed by a Victorian paediatric neurologist.

Infants underwent a single assessment that took 1-2 hours. The assessment involved a detailed interview with the parent or guardian regarding details of the medical history as

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documented in the study assessment case report form (Appendix B), including demographics, family history, pregnancy and perinatal history, details of the epilepsy, development, neurologic and non-neurologic comorbidities, treating doctors and prior investigations. A medical and neurologic examination was performed in living patients. Home video recordings of seizures and non-epileptic episodes were reviewed. This data supplemented and/or clarified that recorded from the medical record as listed in Table 3.3 above.

Further diagnostic investigation was suggested to the treating doctor if a specific aetiology was suggested by the information obtained in the face-to-face assessment or upon review of medical records, EEG, brain imaging or other investigations (e.g. FCD), or if certain routine and appropriate investigations had not been performed or needed repeating e.g.. MRI or EEG.

3.5.6 Genetic testing Research genetic testing was undertaken in infants with SEI of unknown aetiology, following clinical assessment. DNA was obtained from the participant and, where possible, both parents. DNA sources included blood, saliva or fibroblasts. Testing involved multigene panel screening via the massively parallel sequencing techniques of WES and molecular inversion probes (MIPS), as well as targeted single gene testing.

In 2013 and 2014, multigene panel testing using MIPS was the NGS method used. Additionally, some infants had MIPS testing prior to 2013 if they had been recruited to related research studies of my secondary supervisor Professor Ingrid Scheffer. WES commenced in 2015, and essentially replaced MIPS. Thus, some infants had MIPS only, some WES only, and some MIPS followed by WES. Additionally, single gene testing was performed in patients in whom clinical details suggested a particular gene if such testing was performed by research collaborators (e.g. TBC1D24).

MIPS. MIPS multigene panel testing was performed by the Mefford Laboratory at the University of Washington, Seattle, USA, using MIPS as previously described (Carvill, Heavin, et al., 2013; O'Roak et al., 2012). Testing was performed initially on the patient only. The technique used is as follows:

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 MIPS are single-stranded DNA sequences that are designed to capture a ~112-bp stretch of DNA. MIP arms are complementary to the 5’ and 3’ ends of regions of genomic DNA, similar to polymerase chain reaction (PCR) primers, and are connected by a common backbone sequence. MIPs are designed to capture all exons and exon-intron boundaries of genes of interest (multiple MIPS for each gene)  MIPS are pooled for all regions (genes) of interest, and 100 ng of genomic DNA added from a single patient. The MIPS hybridise to genomic DNA and circularize, leaving a gap over the region of interest  Gaps are filled with DNA polymerase and MIPS fully circularized by ligating the ends with ligase  Exonuclease is added to remove linear DNA (from MIPS and genomic DNA)  Patient-specific ‘barcodes’ are introduced during the DNA amplification step which relies on PCR using ‘universal’ primers that bind to the MIPs common backbone. The reverse primers are designed to include the barcode sequence. Individual patient MIPS ‘libraries’ are pooled  Pooled libraries from multiple patients are sequenced using massively parallel sequencing  The sequence reads are sorted by individual patient barcode identifier, mapped to the reference genome, and variants identified  Variants are filtered during analysis based on sequence quality (QUAL>30, QD>5), allele balance (AB>0.7) and population frequency  Non-synonymous, frameshift and splice-site variants are analysed further if they meet quality control criteria, and are absent in control populations for dominantly inherited genes and present in <1% of controls for recessively inherited genes.

Parental DNA was then tested to determine variant segregation using MIPS containing only the sequence of interest. Variants were sequenced to high depth to allow detection of low levels of somatic mosaicism where present. Dominant variants arising de novo, inherited from a mosaic parent or segregating with the disorder were considered pathogenic. Recessive or X-linked variants that segregated appropriately were

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considered pathogenic. Microsatellite testing was performed to confirm maternity and paternity.

Multiple targets of the panel were developed during the study period. The known and candidate genes included in each panel, and the number of infants tested with each target, are listed in Appendix C.

WES. WES was performed by Dr Stefanie Eggers in the Translational Genomics Unit - Research Genomics Service at the MCRI. Analysis of an epilepsy-specific gene panel based on WES data was performed. Dr Eggers also performed a second sequencing technique, PCRbrary, for confirmation and segregation of variants of interest. Variant curation was performed by me with support from Dr Eggers, then variants of interest were discussed at meetings attended by Dr Eggers, me and Prof Ingrid Scheffer to reach a consensus regarding pathogenicity. WES was performed on the patient only.

The concentration and quality of each DNA sample was determined using the Qubit High Sensitivity assay (Invitrogen, Massachusetts, USA) and the TapeStation 2200 Genomic DNA tape and reagents (Agilent Technologies, California, USA). Samples less than 50ng, of a concentration less than 10 ng/ul, or a DNA integrity number less than 6 (highly degraded DNA) were deemed inadequate and DNA recollected.

50ng DNA samples were used for library preparation at a concentration of 10 ng/ul for automated batches using the BRAVO liquid handler (Agilent) or 25 ng/ul for manual preparation (SureSelect QXT Clinical Research Exome kit, Agilent). DNA was fragmented using the transposase-based fragmentation method, and tagged for subsequent low copy amplification. The DNA was then hybridized to the capture library, which consisted of RNA baits containing a biotin-label. Hybridized DNA-RNA baits were captured using magnetic Streptavidin-coated beads (MyONE Streptavidin beads, Invitrogen). Unbound DNA was discarded, the beads washed, and the targeted regions eluted from the beads. Post-capture, the DNA fragments underwent further PCR amplification and each patient’s sample tagged with a unique barcode. Post-capture library quality was assessed using the TapeStation 2200 D1000 tapes and reagents

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(Agilent). Equimolar DNA libraries from multiple patients were pooled and multiplex WES performed on the HiSeq 4000 (Illumina) sequencing system.

Each run was de-multiplexed based on the individual patient barcode and each patient’s reads mapped to the reference genome (Genome reference consortium human genome build hg19). DNA sequence data was imported into the variant filtering program CPipe (Sadedin et al., 2015), a clinical and research pipeline for WES data analysis. All exonic variants in all genes were identified and recorded.

Analysis was confined to a ‘targeted’ WES, with variants in a panel of 341 genes previously reported to cause infantile epilepsies analysed. The gene list is provided in Appendix D. Variant call files obtained from CPipe were filtered for analysis using the program Variant Studio (Illumina) according to the following criteria: 1) variants in 341genes in panel only, 2) frameshift, non-synonymous missense, splice-site, initiator codon, stop-loss, or inframe insertion/deletion variants and 3) variants present at <1% in control populations (ExAC, ESP6500, 1000Genomes (exac.broadinstitute.org , evs.gs.washington.edu/EVS/ , www.internationalgenome.org).

Identified variants were ‘curated’ to determine variants of interest based on 1) whether the gene was consistent with patient’s phenotype, 2) whether zygosity of variant was as expected for disease, 3) variant type, 4) in silico predictions of pathogenicity (PolyPhen2, SIFT, MutationTaster (genetics.bwh.harvard.edu/pph2/ , sift.jcvi.org, www.mutationtaster.org), 5) interspecies conservation of residue, 6) presence of variant in control populations, 7) variant previously reported to be pathogenic in the medical literature or ClinVar disease database (www.ncbi.nlm.nih.gov/clinvar/) and 8) variant quality and coverage.

Variants of interest were considered to be those that were consistent or possibly consistent with the patient’s phenotype, fit the expected zygosity, were present in <1:1000 in control populations for dominantly-inherited genes or <1:100 for recessively inherited genes, and were predicted damaging or possibly damaging by one or more in silico tool. Low quality variants were reviewed in the Integrative Genome Viewer (software.broadinstitute.org/software/igv/) to determine variants that may have been

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called due to sequencing-related errors or alignment errors to the reference genome, and not further examined if misalignment was confirmed.

A second sequencing technique, PCRbrary, was performed on the patient sample to confirm the presence of the variant(s) of interest and on the parental DNA samples to determine segregation. PCR primers spanning the whole exon of interest (intron-intron spanning this exon where possible) were designed, aiming for sequence lengths of approximately 250 base pairs. PCR using these primers was performed to amplify the sequence of interest using 50 ng of DNA. In a second PCR, NGS sequencing adapters were added to the PCR amplicon, and samples tagged with individual barcodes. After each PCR, the amplicons were captured using Streptavidin-coated magnetic beads, and the sample ‘cleaned’ with AMPure (Beckman-Coulter) to remove dNTPs, polymerases, PCR primer dimers and PCR buffer. DNA was eluted from the magnetic beads using water. DNA samples from multiple patients and parents were pooled, hybridized to a flow cell by the attached adaptors and subject to multiplex sequencing using the MiSeq sequencing system (Illumina). Samples were demultiplexed using the individual patient barcode to identify individual samples. Sequenced reads were imported into the MiSeq Reporter program, mapped to the reference genome and variants identified. Variants not confirmed by this independent technique were considered to represent WES sequencing errors.

Following segregation, variants were classified as pathogenic, possibly pathogenic, VOUS or polymorphism based on the variant classification scheme provided in Appendix E.

A small number of infants had WES or WGS performed through other studies in which they were also participating.

Mutations identified through research genetic testing that were considered pathogenic were reported to the participant’s treating doctor, who communicated the results to the parents/guardian. Typically they were asked to have the mutation confirmed through a routine clinical laboratory. Genetic counselling was arranged by the treating doctor.

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3.6 Data documentation

Data from ascertainment sources and review of medical records to determine if inclusion criteria were met was recorded in an ‘ascertainment’ database.

Data obtained from medical record reviews, consensus EEG and MRI reviews, and review of other diagnostic investigations were recorded for all patients with SEI directly into a separate study database. Data from face-to-face assessments were recorded initially on the study case report form (Appendix B), then entered into the study database.

3.7 Analysis of group data

3.7.1 Analysis of search strategies The number of entries searched, medical records reviewed, and infants identified as having SEI were calculated for each search strategy (e.g. EEG, NICU databases). Duplicate identifications were noted within and between ascertainment sources and different search strategies, both to ensure accurate numbers of individual patient identifications and provide validation of the screening processes.

3.7.2 Epidemiology including genetic epidemiology The incidence of SEI was calculated using live birth rates obtained from the Australian Bureau of Statistics (www.abs.gov.au), and presented according to standard epidemiologic convention (per 100,000/year) (Thurman et al., 2011).Population migration rates were used to correct the calculated incidence to account for Victorian- born infants who may have moved out of the state prior to seizure presentation. The incidence of each epileptic syndrome, and that of genetic and non-genetic aetiologies, was also calculated.

3.7.3 Clinical characteristics and aetiologies The number and proportion of infants with the clinical and demographic features listed in Table 3.3 were determined. The chi square (χ2) statistic was used to determine significant differences in the distribution of categorical variables.

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3.7.4 Health economics Current diagnostic pathway

In current clinical practice amongst Victorian paediatric neurologists, diagnostic investigation is performed in ‘tiers’, which are undertaken when the history and examination do not suggest a specific diagnosis. The second and third tier investigations are only performed if first tier investigations are not diagnostic, or do not strongly suggest a diagnosis that allows further targeted testing. All tests in each tier are typically performed concurrently Exceptions include that MRI may be performed separately (prior to or after) the rest of Tier 1, and that skin biopsy may be performed separately to liver and muscle biopsy in Tier 3. Some patients have parts or all of a tier repeated if initial investigations did not find a cause. More recently, a small number of infants have also undergone gene panel testing clinically, this typically performed after Tier 2 testing. Investigations may be repeated over time (most commonly repeat brain imaging which may reveal cortical dysplasia after myelination is complete).

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Table 3.5 Current standard investigations for aetiologic diagnosis of infants with SEI with unknown cause at time of presentation.

Tier Investigations

Tier 1 MRI and MRS brain Urine metabolic screen: organic acids, amino acids, P6C, S-sulphocysteine, guanidinoacetic acid, purines and pyrimidines Blood – biochemistry: FBE, UEC, glucose, Ca/Mg/PO, LFTs, lactate, ammonia, amino acids, acylcarnitines, biotinidase, uric acid Blood – chromosomal microarray Tier 2 Blood – other: karyotype by G-banding, mitochondrial mutations, POLG common mutations, transferrin isoforms, copper and caeruloplasmin, very long chain fatty acids, white cell enzymes CSF – cell count, protein, glucose*, lactate*, pyruvate*, amino acids*, neurotransmitters, MTHFR (* = paired with serum) Repeat imaging MRI brain

Tier 3 Skin biopsy - electron microscopy for changes of neuronal ceroid lipofuscinosis, fibroblast culture (for DNA source) Liver and muscle biopsies – respiratory chain enzyme analysis

Economic evaluation of current and potential future diagnostic pathways

An economic evaluation of the cost and yield of current standard diagnostic testing compared with the cost and yield of WES being added to that pathway, at various points in the pathway, was performed. Additional comparisons were made with diagnostic pathways that did not include Tiers 2 and/or Tier 3 testing.

Approach to costing: The number of tests each infant underwent is large and be time- consuming to individually cost. Thus, the cost of each tier was modelled, and ‘standard’ costs were used instead of costing the tests performed at the individual patient level. That is, each tier cost the same for each child undergoing that tier.

The current (2016) cost to the RCH of each pathology test was used in this evaluation. These costs were provided by the RCH Pathology unit for tests performed internally, and obtained from the relevant providers where tests were performed externally. For other costs of tiers 1, 2 and 3, such as admissions or anaesthetics required to carry out the tests, the 2014-5 hospital costs of undergoing these tests were obtained, as the 2015- 2016 data is not yet available. These costs were corrected for inflation using the Reserve

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Bank of Australia’s Inflation Calculator (www.rba.gov.au/calculator). Admission costs for four or five patients who underwent a particular tier of testing were obtained from the Decision Support Unit (DSU) at RCH. Only the costs relevant to the investigations were considered. For example, the patients undergoing Tier 1 testing whose costs were obtained were infants with new presentations of spasms, whose admissions were both for investigations and for institution of treatment. Additional days of admission for treatment were not included. The mean of the costs for each representative patient was used.

The costs of individual pathology tests are listed in Table 3.6. The sources and costs of groups of pathology tests, and non-pathology-related costs are listed in Table 3.7.

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Table 3.6 Cost of pathology tests

Tier 1

Urine Urine metabolic screen $150 Blood Chromosomal microarray $510.40 FBE $16.95 UEC, Ca/Mg/PO4, glucose, uric acid, LFTs, lactate, ammonia $17.70 Vitamin B12 $23.60 Copper and caeruloplasmin $50.70 Amino acids $120 Acylcarnitines $100 Biotinidase $120 TOTAL TIER 1 $1,109.35

Tier 2 Blood Common mitochondrial DNA mutations and POLG sequencing $1200 Transferrin isoforms $140 Very long chain fatty acids $250 White cell enzymes $474 CSF Cell count and differential, protein, glucose, lactate $71 Pyruvate $50 Amino acids $120 Neurotransmitters $100 TOTAL TIER 2 $2,405

Tier 3 Liver Respiratory chain enzyme analysis $1,130 Histology $97.50 Muscle Respiratory chain enzyme analysis $1,130 Histology $370 Skin Fibroblast culture $208.74 Electron microscopy $97.50 TOTAL TIER 3 $3033.74

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Table 3.7 Costs of tiers of tests in the diagnostic pathway

Investigation Uncorrected 2016 Source of cost costs costs*

Tier 1

Neurology $819.13 $839.66 DSU, mean cost of neurology unit medical costs for a 2- unit medical day inpatient admission to the neurology ward at RCH costs based on four representative patients (cost for one day = $409.56) Ward and $647.84 $664.08 DSU, mean cost of ward and nursing costs for a 2-day nursing costs inpatient admission to the neurology ward at RCH based on four representative patients (cost for one day = $323.92) Theatre costs $1029.38 $1055.18 DSU, mean theatre costs based on two representative (for GA for patients (NB only two patients used as costs were MRI. Includes clearly falsely low for two other patients) anaesthetist, theatre nurse, theatre technician) Pharmacy $22.46 $23.02 DSU, mean pharmacy costs for a general anaesthetic (taken from the mean costs for four representative patients having a GA for Tier 3 investigations as that is the only pharmacy cost for those patients (those admitted for Tier 1 testing also have pharmacy costs related to their antiepileptic drug treatment which cannot be separated from anaesthetic pharmacy costs) MRI brain $608.00 $608.00 Estimate of hospital cost for imaging, technologist time imaging scanning and radiologist reporting time; is equivalent to (performed the MBS rebate for this test +50% (because the scan under GA as time is 45 minutes and the MBS rebate is for 30 minutes an inpatient) of imaging). Pathology $1109.35 $1109.35 Obtained from RCH Pathology and VCGS, based on costs to the RCH for these investigations TOTAL TIER 1 $4299.29

Tier 2 Neurology $294.02 $301.39 DSU, mean cost of neurology unit medical costs for a unit medical day admission to the Day Medical Unit at RCH to costs undergo these investigations based on four representative patients Ward and $215.66 $221.06 DSU, mean cost of ward and nursing costs for a day nursing costs admission to the Day Medical Unit at RCH to undergo these investigations based on four representative patients Pharmacy $4.19 $4.30 DSU, mean pharmacy costs for sedative medication given to perform Tier 2 investigations based on four representative patients Pathology $2405.00 $2405.00 Obtained from RCH Pathology and VCGS, based on costs to the RCH for these investigations

TOTAL TIER 2 $2931.75

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Repeat MRI $2100 $2100 DSU, cost estimate for epilepsy patients admitted (day admission to medical imaging at RCH) for 3T MRI brain imaging under GA Tier 3 Neurology $532.43 $545.77 DSU, neurology unit medical costs for a 1.3-day unit medical inpatient admission to the neurology ward at RCH (taken costs from mean costs for four representative patients undergoing Tier 1 testing, cost for one day = $409.56) Ward and $421.10 $431.65 DSU, cost of ward and nursing costs for a 1.3-day nursing costs inpatient admission to the neurology ward at RCH (taken from mean costs for four representative patients undergoing Tier 1 testing, cost for one day = $409.56, cost for one day = $323.92) Surgeon costs $941.73 $965.33 DSU, double the mean surgeon costs for five representative patients undergoing muscle biopsies (surgeon cost is related to operating time; Tier 3 involves muscle and liver biopsies which takes approximately twice as long as muscle biopsy only) Theatre costs $2074.45 $2126.44 DSU, based on mean theatre costs for five (includes representative patients undergoing muscle biopsies anaesthetist, (theatre cost is not related to operating time, so costs theatre nurse, were not doubled like the surgeon cost was) theatre technician) Pharmacy $22.46 $23.02 DSU, mean pharmacy costs for a GA based on five representative patients undergoing muscle biopsies Pathology $3033.74 $3033.74 Obtained from RCH Pathology, VCGS and the SNPS, based on costs to the RCH for these investigations

TOTAL TIER 3 $7125.95

WES gene $2200 $2200 Dr Damien Bruno, Laboratory Director at VCGS , panel hospital cost of commercial WES gene panel performed at VCGS (sequencing, analysis and clinical report) DSU = Decision Support Unit, Royal Children’s Hospital, GA = general anaesthetic, MBS = Medicare Benefit Scheme, MBS rebate = fee paid by the Federal Government through the Medicare Benefit Scheme to the provider for a particular medical service, to offset the cost of the service incurred by patients or service providers, VCGS = Victorian Clinical Genetics Service, SNPS = State Neuropathology Service at the Alfred Hospital, Melbourne

* The 2016 costs were used in the costing analysis. Costs were corrected for inflation where necessary. Admission costs obtained from the DSU are based on costs from the 2014-2015 financial year. Those costs were corrected for inflation using the Reserve Bank of Australia Inflation Calculator http://www.rba.gov.au/calculator/. As per the calculator, these costs increased by 2.5% over two years (2014-2015 to 2016-2017), at an average annual inflation rate of 1.2%. Pathology, imaging and WES costs (including all costs associated with repeat MRI) were based on current (2016-2017 financial year) costs.

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Currently, our institute is establishing clinical multigene testing as WES, with a gene panel analysis. For consistency of costing and usefulness of this data at our institution, all diagnoses made on multigene testing techniques performed on infants in this study (clinical gene panel, MIPS, gene panel from WES data) were considered to have been made with one technique – WES gene panel. The cost was determined to be $2200, based on the planned commercial charge for WES gene panel testing (source: Dr Damien Bruno, Laboratory Director, VCGS).

Economic evaluation method: Similar to the approach for costing, a standardized approach to which patients underwent which tiers of testing was used rather than using the tiers actually performed for each patient. This approach was chosen to see how the different diagnostic pathways performed in an ideal scenario, and leaves aside any issue of clinician preference and choice in how their patients are investigated. Thus, it was assumed that diagnostic investigation in all infants followed the prescribed pathways and that the diagnoses were made at the earliest possible point in the pathway at which they could reasonably have been made.

The most relevant example of the latter point is that all infants with brain malformations clearly visible (at our research imaging review) on the first MRI were considered to have had a diagnosis made on Tier 1 testing (even if the diagnosis was not actually made until later clinical or research review). Infants with a brain malformation not visible, or suspected but not clearly diagnosed, on the first scan but clearly visible at a later time point were considered to have had their diagnosis made on their repeat MRI.

For diagnoses suspected at particular points in the diagnostic pathway that required targeted testing to confirm the diagnosis, the costs of targeted testing were added to the total costs. Examples of targeted genetic testing included SCN1A testing in infants with Dravet syndrome and single gene testing in particular dysmorphic syndromes. In this model, most targeted gene testing would have been performed immediately after Tier 1 testing as many of these diagnoses can be suspected early in the course of the condition. Other examples of targeted testing included tissue biopsies (i.e. Tier 3 testing performed earlier in the diagnostic pathway) in infants with suspected mitochondrial disorders. In infants with a mitochondrial disorder, the diagnosis was considered ‘suspected’ after

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Tier 2 testing revealed elevated CSF lactate. In reality, mitochondrial disorders were suspected in these infants after Tier 1 when the serum lactate was elevated, MRI brain showed signal abnormalities and MRS showed a lactate peak. However, given the possibility of spurious or transiently elevated serum lactates in infants without a mitochondrial disorder, and that signal change (and sometimes elevated CSF lactate) can be seen in other disorders, those Tier 1 findings were not considered to constitute a suspected mitochondrial disorder here.

The costs used for genetic and other confirmatory testing in particular infants are listed in Table 3.8. It should be noted that there was large variability in the cost charged for sequencing of different genes. The costs used were the 2016 costs charged for the relevant test by the laboratory that performed the test in that infant (where such information was available). For one infant (who had NSD1 sequencing), it was not known where the test was performed. Thus, the cost charged by a large genetic testing company, GeneDx (http://www.genedx.com/) was used. For two infants, whose confirmatory tests were biopsies of muscle, liver and skin for histology and respiratory chain enzyme analysis, the cost of Tier 3 testing was used. If WES was immediately after the tier in which a diagnosis was suspected, it was considered that that diagnosis would be confirmed on WES rather than targeted gene testing, thus the costs of targeted testing were removed in that instance. For example, if an infant had Dravet syndrome suspected at the time of Tier 1 testing, and WES was the second step in the pathway (as in scenarios 5-7 below), WES would be used to confirm that diagnosis rather than specific SCN1A sequencing. In contrast, if WES was at step 3 or later (as in scenarios 2- 4 below), SCN1A sequencing would be performed at that point and the diagnosis considered to be confirmed on Tier 1 testing.

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Table 3.8 Costs of confirmatory testing of suspected diagnoses

Test Cost Source

SCN1A sequencing $875 West of Scotland Genetic Service, Glasgow

TBC1D24 sequencing $120 Dr Mark Corbett, University of Adelaide, Adelaide NSD1 sequencing $4,061 GeneDx (www.genedx.com)

Aicardi-Goutières gene panel $4,714 GeneDx (www.genedx.com)

PNPO sequencing $1,101 Metabolic Unit, VU University Medical Centre, Amsterdam Tier 3 testing $7,025.91 As per Methods chapter

Costs are listed in 2016 Australian dollars.

The costs of confirmatory genetic testing in infants whose aetiologic diagnosis was clear on another test (e.g. TSC1 and TSC2 testing for infants whose MRI is diagnostic of TS) were not included in this analysis.

The cost of diagnostic investigation in infants with a known diagnosis prior to epilepsy onset were not considered in this analysis.

Thus, the approach taken:

 used the actual diagnoses of infants in this cohort who did not have an aetiologic diagnosis at epilepsy onset  assumed that these diagnoses were made at the earliest point in the modelled diagnostic pathways that they could have been (rather than the point at which the diagnosis was actually made)  assumed that infants progressed through the pathway in a prescribed manner (with none of the variations that occurred in clinical practice, other than performing targeted testing if a particular diagnosis was highly likely)and  assumed that the cost of each tier was the same for each infant (rather than using the actual costs accrued by each infant).

A number of diagnostic pathways were modelled. Each scenario was modelled twice: once assuming that all infants progressed through the pathway until a diagnosis was made (Table 3.9), and the other assuming that infants had no further investigations if

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their seizures were controlled (Table 3.10). The only exception to the previous sentence was that Tier 3 testing was not performed in infants who were strongly suspected to have an occult brain malformation (or if seizures had ceased for the scenarios in which all infants progressed through the diagnostic pathway) as these infants would not typically undergo tissue biopsies in clinical practice.

The latter approach takes into account that, in real-world practice, some infants will not be further investigated if seizure have ceased. For the second approach, real patient data regarding ongoing seizures was used. Step 2 was only performed if seizures were ongoing for >1 month after presentation, step 3 if seizures were ongoing >3 months and steps 4 and 5 if seizures were ongoing >6 months. Deceased patients were assumed to have had the whole pathway performed in this scenario, even if seizures were not ongoing or if death occurred earlier than six months after presentation (as, in clinical practice, many of these infants have the later tier tests performed pre- or peri-mortem if they have not already been done).

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Table 3.9 Simulated diagnostic pathways modelled in this economic evaluation assuming that infants progressed through the pathway until an aetiologic diagnosis was made

Scenario Step 1 Step 2 Step 3 Step 4 Step 5 1 Tier 1 Tier 2 Repeat MRI Tier 3* -

2 Tier 1 Tier 2 Repeat MRI Tier 3* WES

3 Tier 1 Tier 2 Repeat MRI WES Tier 3*

4 Tier 1 Tier 2 WES Repeat MRI Tier 3*

5 Tier 1 WES Tier 2 Repeat MRI Tier 3*

6 Tier 1 WES Repeat MRI Tier 2 -

7 Tier 1 WES Repeat MRI - -

*Tier 3 not performed if a brain malformation was strongly suspected or if seizures had ceased as these infants would not typically undergo tissue biopsies in clinical practice

Table 3.10 Simulated diagnostic pathways modelled in this economic evaluation assuming that infants progressed through the pathway until an aetiologic diagnosis was made only if seizures were ongoing

Scenario Step 1 Step 2 only if Step 3 only if Step 4 only if Step 5 only if seizures ongoing seizures ongoing seizures ongoing seizures ongoing >1 month >3 months >6 months >6 months 1 Tier 1 Tier 2 Repeat MRI Tier 3* -

2 Tier 1 Tier 2 Repeat MRI Tier 3* WES

3 Tier 1 Tier 2 Repeat MRI WES Tier 3*

4 Tier 1 Tier 2 WES Repeat MRI Tier 3*

5 Tier 1 WES Tier 2 Repeat MRI Tier 3*

6 Tier 1 WES Repeat MRI Tier 2 -

7 Tier 1 WES Repeat MRI - -

*Tier 3 not performed if a brain malformation was strongly suspected as these infants would not typically undergo tissue biopsies in clinical practice

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Analysis of economic costs: The following analyses were performed across the group for both the scenario in which all infants continue the investigation pathway until a diagnosis is reached, and for the scenario in which investigation ceases if seizures have stopped:

 The number of diagnoses made and yield of each tier in each diagnostic pathway used  Diagnostic yield, cost per diagnosis and cost per incremental diagnosis for each diagnostic pathway used  The additional costs incurred by WES and costs saved from tiers in the current pathway for each diagnostic pathway used.

3.7.5 Study time frames This PhD study was conducted during the four years 2013-2016, this period incorporating six months of maternity leave.

Infants with SEI born in the three years 2011-2013 were ascertained according to the following time frames:

 Reports of EEGs performed between January 1 2011 and December 31 2015 were reviewed. The latest possible presentation for a patient born in 2013 was June 30th 2015 (point at which a child born on 31 Dec 2013 would be 18 months old). Records were screened for an additional six months to identify any infants with delayed presentations to medical care.  NICU databases were searched from January 1 2011 to December 31 2013 to capture all neonates with seizures born in 2011-2013, as database entries are listed by date of birth.  Neurologist notifications of potential participants occurred only within the PhD timeframe of 2013-2015.

These strategies were performed retrospectively to identify patients presenting in 2011- 2012 and at intervals to identify patients presenting in 2013-2015 in a (quasi-) prospective manner.

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Assessment of infants with SEI (including genetic testing) occurred throughout the study period.

Table 3.11 Study time frames

Year 2011 2012 2013 2014 2015 2016

PhD time frame

Year of birth

Year of presentation

EEG data review period

NICU database review period

Neurologist notification period

Assessment of infants with SEI

3.8 Ethics approvals

Human Research Ethics Committee approval was sought and obtained for the study. The two study components have distinct ethical issues.

Ascertainment involved searching EEG reports, NICU databases and medical records, and contacting treating doctors, at multiple sites without patient contact and consent as noted in the Ascertainment section above, in order to identify all infants with SEI who met inclusion criteria. An ethics application, including a waiver of consent, was required for each hospital involved in the study, namely RCH, MMC, Austin, RWH and MHW. Applications to waive the requirement for patient consent for a study performed by researchers who do not have rightful access to those patients’ medical records must meet the criteria outlined in Section 2.3.6 of the National Statement on Ethical Conduct in Human Research document from the National Health and Medical Research Council (www.nhmrc.gov.au/guidelines-publications/e72). For this study, the relevant Ethics Committees were satisfied that the criteria were met and approved the waivers (RCH – study number 32288, MMC study number 13274B, Austin study number H2013/05082, RWH project 13/24, MHW study number R13-45, see Appendix F for the criteria for approval of a Waiver of Consent as they relate to this study, and Appendix G for study ethics approval documents). Patient contact and consent for the purposes of

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ascertainment was only sought where further detail, not available in the medical records or from treating doctors, was required to determine whether inclusion criteria were met.

Site-specific consent was not obtained to search EEGs performed at Victorian regional EEG laboratories. Rather, neurologist referral was considered the screening source for these sites, each site having a neurologist, who had rightful access to this data, as they were responsible for reporting EEGs. Reporting neurologists were asked to notify me of infants whose EEG suggested a possible SEI. For these infants, deidentified clinical information was provided to me to determine whether the infant possibly met inclusion criteria. Date of birth was provided to cross-reference infants with possible SEI with those already identified at hospital sites. Consent to contact the families of infants (not already identified) was then sought to clarify whether inclusion criteria were met. The process involved the reporting neurologist contacting the treating doctor to seek consent from the family to be contacted about the study. The Parent Information and Consent Form (PICF) and covering letter were given to families who agreed to be contacted by their treating doctor, the process of recruitment then proceeding in the same manner as for patients identified through Victorian hospital sites. I was provided with identifying clinical information (name, contact details) only once a family agreed to be contacted. Families who did not agree to contact were not further pursued. In fact, no families declined contact. Thus, there were no issues with ensuring epidemiologic accuracy of deidentified screening sources, as clinical information was made available for all infants with possible SEI, allowing determination of 1) novel or duplicate identifications and 2) which infants had a definite SEI. The reporting neurologist was also asked to determine the number of EEGs performed on children less than two years old at each site in the study time frame, to allow accurate documentation of the total number of EEGs searched.

The assessment component of the study involved patient contact and consent to undergo a face-to-face assessment and, in some cases, genetic testing. This component of the study was based at RCH and covered by RCH ethics approval (study number 32288) via a Standard Research (non-drug/device) application. Two versions of the PICF and cover letters were available, one for living and one for deceased patients. The Plain Language Advisor assisted in writing the PICF and cover letter for deceased patients.

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The process for seeking consent varied slightly from site to site depending on that hospital’s Ethics Committee requirements. However, the typical process was:

1. The infant’s treating doctor was contacted to notify of their patient’s potential eligibility for the study, and to identify any reasons that recruitment might not be appropriate (e.g. recent death, difficult social circumstances etc.). 2. Treating clinician contacted the parents/guardians of eligible infants to seek permission for the principal investigator to provide information on the study.

a. Where permission was denied, no further contact was made. b. Where permission was given, the principal investigator sent out study information and/or called the family to discuss the study. Separate PICFs were provided for living and deceased infants (Appendix 8).

3. Informed consent was obtained on those who agreed to participate in the study.

Ethics approval for this phase was not required at other study sites, although MMC required that patients in whom recruitment was sought be first contacted about the study via a letter from the relevant MMC Head of Department (usually Paediatric Neurology), which advised them why they had been contacted and how they had been identified by non-MMC investigators, as well as provided information about the study and the method of contacting the study investigators if they were interested in participating.

Consent was waived for recording re-identifiable data for the study on infants with well- characterised severe epilepsy who did not undergo face-to-face assessment.

Yearly progress reports and a final report upon study completion were required and provided to each study site.

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Chapter 4: Ascertainment and epidemiology

4.1 Ascertainment

Ascertainment strategies identified 114 infants with SEI who met clinical and demographic inclusion criteria. The process of ascertainment and confirmation of SEI diagnosis is detailed below and in Figures 4.1 and 4.2.

4.1.1 Ascertainment sources, search strategies and confirmation of SEI diagnosis EEG databases

Source: 4505 EEGs were performed in Victoria in 2011-2015 in children younger than 2 years old. The number of EEGs and reporting neurologists at each site are listed in Table 4.1. The two tertiary paediatric hospitals, RCH and MMC, performed 84% of the EEGs. There was variability in the number of EEGs performed per year at MMC and Geelong due to periods of extended leave taken by the neurologist or technologist, and some regional sites ceased EEGs on infants.

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Table 4.1 Number of EEGs performed on children less than two years old by site and year of EEG

Number of EEGs

2011 2012 2013 2014 2015 Total Notes

RCH 517 493 483 585 552 2630

MMC 239 235 235 204 243 1156 Fewer EEGs in 2014 due to neurologist extended leave Austin 62 92 84 73 60 371 Geelong 35 21 20 5 12 93 Infrequent EEGs on <2yos from Jan 2014 to September 2015 due to technologist extended leave Bendigo 20 16 20 22 6 84 EEGs on <2yos ceased in 2015 (occasional study done) due to inadequate quality recordings Ballarat 23 34 24 29 17 127

Shepparton 0 0 5 8 12 25 EEGs began at this site February 2013

Warrugal/ 6 10 3 0 0 19 EEGs ceased at this site in April Frankston 2013 due to laboratory closing

Total 902 901 874 926 902 4505

Search strategy: 4501 available EEG reports were reviewed. Reports were deidentified (name removed) for 255 EEGs performed at Bendigo, Ballarat, Shepparton and Frankston/Warrugal EEG laboratories, but dates of birth were available. EEG recordings were reviewed for four EEGs that had no report available.

3290 children younger than 2 years old had one or more EEG (range 1-19, median 1) during the years 2011-2015. 577 children had more than one EEG. 121 children had EEGs at more than one site, and 203 in more than one calendar year.

Review of referral clinical details and EEG findings available in EEG reports excluded 2695 children. 1294 were born outside 2011-2013. 1401 did not have SEI; they had FS (145), seizures but not SEI (181), episodes not clearly seizures (955), and episodes that were not seizures (120).

After EEG report review, 595 children had possible SEI and underwent medical record review to determine if they met clinical and demographic inclusion criteria.

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Confirmation of diagnosis: Medical record review identified 112/595 children who had SEI meeting clinical and demographic inclusion criteria. 457 children were excluded following medical record review; 434 did not have SEI (FS in 11, seizures but not SEI in 210, episodes not clearly seizures in 175, episodes that were not seizures in 38), and 23 had SEI but were born outside Victoria (12 in other states of Australia, 11 overseas). For 27 children, medical record review did not clarify whether inclusion criteria were satisfied. Of these, 24 were ultimately excluded and 3 included. For the 24 who were excluded, it was determined that inclusion criteria were not satisfied via review of the EEG recording in six (EEG not epileptiform; reported variants were physiologic transients and non-specific sharp waves), discussion with the treating doctor in 14 (no hospital medical record in one, insufficient information in medical record in 13), and face-to-face assessment in four. Clinical information for the three included patients was determined by phone interview with a parent in one, and by discussion with the treating doctor and my PhD supervisor, Simon Harvey, in two2.

Therefore, in total, 114 children had confirmed SEI from search of EEG databases, comprising 3.5% (114/3290) of all children <2 years old who had an EEG. Of these infants, 51 (45%) were identified in more than one year, and 33 (29%) at more than one site. 95 (83%) were identified at RCH, 32 (28%) at MMC, 12 (11%) at Austin, 2 at Geelong, 1 at Bendigo, 2 at Ballarat and 1 at Shepparton. No infants were identified at Frankston/Warrugal. Just one infant was not identified at either RCH or MMC, having had EEGs only at Ballarat. This infant was, however, identified by neurologist referral also, following outpatient appointments at RCH and Ballarat.

2 These two infants did not technically meet treatment resistance inclusion criteria, but a decision was made to include them as it was felt that they almost certainly would have if additional treatment was given. One neonate with an EIEE-like epilepsy and absent respiratory drive underwent palliative treatment with a decision made to give no additional antiepileptic medication. The other infant had frequent tonic seizures with a markedly abnormal EEG, but the family failed to attend further appointments and, thus, the infant received only one antiepileptic drug.

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Figure 4.1 Flow diagram showing the process of identifying infants with SEI from EEG reports

* Reviewed EEG reports for 4501 EEGs and recordings for 4 EEGs for which report not available ^ 120 not seizures, 955 unclear if seizures, 145 febrile seizures, 181 seizures but not SEI ~ 38 not seizures, 175 unclear if seizures, 11 febrile seizures, 210 seizures but not SEI

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% by review of EEG recording in 6, discussion with treating doctor or family in 21 # 1 not seizures, 8 unclear if seizures, 15 seizures but not SEI Neonatal Intensive Care Unit databases

Source: There were 379 NICU database entries that noted ‘seizures’ in neonates born during 2011-2013. The number of entries across the state was similar from year-to-year.

Table 4.2 Number of NICU database entries listing ‘seizures’ by site and year of EEG

Number of database entries

Site 2011 2012 2013 Total

RCH 45 48 33 126

RWH 27 28 35 90

MHW 30 28 35 93

MMC 21 27 22 70

Total 123 131 125 379

RCH = Royal Children’s Hospital Melbourne, RWH = Royal Women’s Hospital, MHW = Mercy Hospital for Women, MMC = Monash Health

Search strategy: In total, 376 neonates had one or more database entry. Three neonates were identified twice (each at two sites), all three having been transferred to RCH for further management. Medical records were available for review in all neonates and were sufficient to determine inclusion or exclusion in all.

Confirmation of diagnosis: Medical record review excluded 364 neonates. 247 had seizures but not SEI, of whom 215 had acute symptomatic seizures including HIE in 117, stroke in 30, haemorrhage in 26, central nervous system infection in 29 and hypoglycaemia in 10. 76 had episodes that were not clearly seizures and 42 had episodes that were not seizures.

Medical record review identified 11 neonates with confirmed SEI in the neonatal period; two were identified at two sites. Seven were identified at RCH, two at RWH, three at MWH and two at MMC.

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135 neonates had an EEG performed while in the NICU. All 11 neonates with SEI identified from NICU databases were also identified from review of EEG reports.

A further 12 infants with a NICU database entry, who did not have SEI in the neonatal period, had confirmed SEI later in infancy. This included eight with acute symptomatic seizures, two with seizures but not SEI and two with episodes that were not clearly seizures in the neonatal period. All 12 were also identified on EEG report review.

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Figure 4.2 Flow diagram showing the process of identifying infants with SEI from NICU database entries

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Neurologist referral

Source: Forty-three infants were referred to the study by Victorian neurologists. All referred infants were discussed with the treating neurologist. It should be noted that neurologist referral to the study was ad hoc and expected to provide duplicate ascertainment, established as a means of capturing rural infants or infants who had moved back from interstate or overseas who may not have been identified through the other screening sources.

Confirmation of diagnosis: Five infants did not meet inclusion criteria. This was determined by review of records and discussion with the neurologist in three patients, and by face-to-face assessment in two. One did not have an epileptiform EEG before 18 months old, two did not meet treatment-resistance criteria, one had inadequate seizure frequency and one was born outside Victoria. The remaining 39 infants met inclusion criteria. All were also identified from EEG reports, including from EEGs at RCH or MMC in all but one (who had EEGs only at Ballarat, as noted above in EEG report section).

Duplicate identification

114 infants met inclusion criteria, 47 being identified through more than one ascertainment source. However, if an infant having an EEG at more than one site (e.g. at RCH and MMC) or in more than one year (e.g. in 2011 and 2012) was considered to have been identified through two different sources, this number increased to 79/114. A further 27 infants had multiple EEGs at only 1 source and in only 1 calendar year. Eight infants were identified through a single EEG only (two of whom had other EEGs performed at laboratories overseas, one of whom was identified in Victoria prior to going overseas). Thus, 107/114 (94%) of infants were identified more than once.

4.1.2 Discussion Analysis of search strategies and inclusion criteria

It was expected that the search strategies and process of confirmation of SEI diagnosis used in this study would provide complete ascertainment of all infants who met the

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study inclusion criteria, with the exception of Victorian-born infants who moved out of the state prior to SEI onset or presentation, a number which was expected to be small.

Possible underascertainment: While the study design was not based on a birth cohort, and therefore by design more prone to incomplete ascertainment, three factors mean that there would likely be minimal difference in the incidence figures derived in this study compared with that from a birth cohort.

Firstly, SEI are such severe conditions that it is highly unlikely that an infant with SEI would not present to medical attention. It is useful to consider the mode and timing of presentation of infants with the two most common seizure types, focal seizures and epileptic spasms. Most infants with focal seizures presented to emergency departments early in the course of their epilepsy, many after the first seizure. The presentation of infants with epileptic spasms to medical attention was more variable with respect to both site and time of presentation, but those who did not present to an emergency department typically presented to a paediatrician either because of the spasms, or developmental regression. The knowledge of the concerning nature of possible epileptic spasms among Victorian paediatricians is good, and is such that infants with possible spasms are typically referred urgently for an EEG. Many more infants were referred for an EEG for this indication than were ultimately diagnosed with spasms (these infants typically having alternative non-epileptic diagnoses such as shuddering), suggesting over-awareness rather than under-awareness of this seizure type. In some cases though, spasms were not promptly recognized, or recognized at all, either due to delayed presentation to medical attention or lack of recognition of spasms by families, or by failure to diagnose the episodes in question as spasms by medical staff. Thus it is not possible to exclude the possibility that infant(s) with spasms (particularly if subtle and self-resolving) did not present to medical attention and were therefore not ascertained.

Secondly, all infants with suspected afebrile seizures of any type are referred for an EEG. There are a limited number of EEG laboratories in Victoria that perform EEGs on children less than two years old. Thus, it was feasible to search EEG reports from all Victorian laboratories and identify all such infants.

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Thirdly, it was expected that most, if not all, infants with SEI would be known to a Victorian paediatric neurologist. Victorian GPs typically refer infants with suspected seizures to a paediatrician or a paediatric neurologist. While some infants with seizures may be managed by a paediatrician, typical practice in Victoria is that any infant with refractory seizures or infantile spasms be referred to a paediatric neurologist. This assumption was borne out; all 114 infants with SEI were seen by a paediatric neurologist. While the rate of neurologist referral to the study was relatively low, given it is usual practice that all infants undergo an EEG, it is unlikely that any infant seen by a paediatric neurologist with SEI would not have been ascertained.

Thus, provided an infant presented to medical attention, it was highly likely that they would be identified by either or both of the EEG and paediatric neurologist screening sources. Two possible exceptions to this were neonates in NICUs, and infants who moved out of Victoria.

In Victoria, all neonates with suspected seizures, except where obviously symptomatic of a metabolic disturbance such as hypoglycaemia and responsive to initial treatment, are transferred to a tertiary NICU. This practice was confirmed by neonatologists staffing the Victorian Neonatal Emergency Transport Service and two of the major secondary neonatal units. Not all neonates with suspected seizures in Victorian tertiary NICUs undergo an EEG (135/376 in this study). Many neonates did not have a conventional EEG, due to the widespread use of amplitude-integrated EEG in Victorian NICUs, which provides a 2-channel EEG recording to be undertaken in the NICU by neonatal staff and is often used for prolonged studies. NICU database searches were therefore performed to account for the possibility that neonates with SEI may be missed by the EEG screening source.

Database searches at all sites identified who did not have seizures, as the seizures field would be coded as yes if the admission diagnosis was seizures or suspected seizures, even if the episodes were subsequently shown to not be seizures. Searching discharge summaries at MMC using the keyword ‘seizures’ also identified the phrase ‘not seizures’. It is our clinical experience that most transient episodes of unusual or abnormal movements, altered awareness or altered autonomic function without an

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obvious alternative cause are considered to be possible seizures in neonates, and would therefore be captured by the database searches. Thus, typical clinical practice and the set-up of these databases meant that searches would over- rather than under-identify infants with seizures. The 247 neonates with seizures that were not SEI clearly did not meet inclusion criteria – 215 had acute symptomatic seizures, and the 32 other infants did not meet treatment resistance criteria (30 had 0 or 1 AED, two had 2 AEDs with no ongoing seizures after the second AED). None of the 12 infants identified on the neonatal databases who had a diagnosis of SEI made later in infancy were thought in retrospect to have met inclusion criteria neonatally. The concern that neonates in the NICU meeting the inclusion criteria may not be identified by EEG was not founded – all 11 with SEI in the neonatal period were identified on both sources. Thus, it is not likely that neonates in Victorian NICUs with SEI were not identified.

The study was not set up to identify infants with SEI who moved interstate or overseas before presenting to medical attention. Thus, some such infants may have been missed unless they moved back to Victoria in infancy and were identified at that point. This was the case for one infant. It was not possible to identify any remaining infants, so a correction for outward population migration was applied to incidence figures to account for this (see below). It should be noted that the figure obtained by correcting for population migration will likely overestimate the number of infants with SEI who moved out of Victoria and were not identified, as two infants who moved out of Victoria (the infant above and another who moved interstate after presentation with SEI) were ascertained.

While each of the above factors has been considered qualitatively in determining possible sources and magnitude of underascertainment, the more formal and quantitative method for calculating completeness of ascertainment where multiple ascertainment sources are used is the capture-recapture method (Posada de la Paz, Groft, & SpringerLink (Online service), 2010). This method works on the assumption that the probability of a case being identified with all ascertainment sources is equal to the probability of a case being identified with one source. That is, that complete identification can be assumed if all cases are identified by all sources independently. While multiple ascertainment sources were used in this study, and the capture-recapture

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method could therefore be applied, the basic assumptions of this method are not valid here. Namely, it is not expected that all infants would be identified by all sources. It was expected that EEG would identify almost all infants (except neonates as not all were referred for an EEG), but that NICU databases (not all infants present in the neonatal period) and paediatric neurologist referral would not (this source was not systematic). However, 94% of infants were identified more than once, which supports a high rate of case ascertainment.

Finally, the possibility that an infant with SEI appeared in a screening source but was erroneously considered to not have SEI needs to be considered. However, the completeness of medical record data available for review, including data from multiple sources (providing data up to the age of two years for most infants with possible SEI on EEG report review) and sites in many patients, minimizes this possibility.

Possible overascertainment: The possibility that an infant deemed to meet inclusion criteria actually did not have SEI also needs to be considered. There are two main ways in which this could occur – if an infant had non-epileptic episodes that were documented in EEG referral clinical detail or medical records as seizures, or if the information on seizure frequency or AEDs available on an infant with seizures was inaccurate.

Confirmation, or evidence strongly supportive, of seizures in almost all infants means that the possibility of the former is low. 80/114 (70%) infants had seizures recorded on EEG, confirming the clinical suspicion of seizures, and a further 9 infants had video (home video in 8, EEG video without simultaneous EEG recording in 1) of episodes reviewed by a neurologist and deemed to be seizures. All but one of the remaining infants had EEG findings consistent with the reported seizure semiology (e.g. hypsarrhythmic EEG in association with spasms)3. Four infants with possible SEI

3 One infant with Aicardi-Goutieres syndrome and severe dystonia was reported to be having frequent seizures with a mix of tonic and clonic features, which were ongoing despite four AEDs. No video of the episodes was available, and there was no contemporaneous EEG. However, an EEG performed a few months prior was markedly abnormal with frequent epileptiform discharges. Thus, while the possibility

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underwent face-to-face assessment after review of medical records and EEG, and discussion with the treating doctor was unable to clarify whether inclusion criteria were met. For two of these four, it was determined that most, if not all, of the episodes thought to be seizures were not seizures4. That the design of the study included the ability to conduct face-to-face assessments has therefore reduced the possibility of inaccurate inclusion in the cohort.

It is also possible, though not likely, that an infant with seizures but not SEI could have been erroneously deemed to have SEI. This possibility was minimized by the detailed medical record and EEG data available on infants with possible SEI, and the ability to obtain further information from paediatric neurologists and families regarding seizure frequency and AED use where required.

Retrospective vs prospective ascertainment: Prospective ascertainment is preferred over retrospective ascertainment, being less prone to selection and classification bias. In this study, sources were screened retrospectively for infants presenting in 2011-2012 and quasi-prospectively (sources searched at frequent intervals) for 2013-2015 (2013 only for NICU databases). Relative to the number of live births, the number of infants with SEI identified in each individual year was similar (see Incidence of SEI section). Further, there was no significant difficulty in obtaining sufficient information to determine inclusion on the infants presenting in 2011-2012 who were identified retrospectively, due to the large volume of historical records available on each infant, as well as a high rate of ongoing management by a paediatric neurologist. Therefore, it does not seem likely that timing of ascertainment relative to presentation has had a significant impact on completeness or accuracy of identification of infants with SEI.

Summary: While potentially inferior to a birth cohort for epidemiologic purposes, the design of this study was chosen for pragmatic reasons, including the prohibitive time, cost and manpower required to develop a birth cohort. While, as discussed above, there

that the episodes in question were not seizures remains in this case, it was deemed that these were more likely seizures than not, and this infant was included. 4 Episodes in one infant were non-pathologic episodes, including staring and flushing, and in the other were paroxysmal episodes consistent with those of alternating hemiplegia of childhood.

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are many potential sources of incomplete ascertainment with non-birth cohort studies, the severity of SEI and the set-up of the medical system in Victoria including the universal application of EEG in this setting minimize the risk of incomplete ascertainment in this instance. Overall, it is likely that all infants with SEI born during 2011-2013 were identified, apart from infants who moved out of Victoria.

Infants with epilepsy not meeting SEI criteria

While the focus of this study was severe epilepsies of infancy, given the absence of a widely-accepted definition of a ‘severe’ infantile epilepsy, and the use of a pragmatically-chosen but novel definition in this study, it was important to consider the group of infants with epilepsy that was ‘not SEI’. More specifically, it was important to understand whether the composition of the ‘not SEI’ group was as anticipated and, in particular, whether some infants whose epilepsy may be considered ‘severe’ by other definitions, such as some with Dravet syndrome, were excluded as was expected.

Data from EEG screening source: Review of the data from the EEG screening source revealed 119 infants with epilepsy onset under 18 months old that did not meet clinical criteria for SEI. It is not known whether all 119 infants met demographic criteria; it is possible that some were non-Victorian-born infants in this group as their place of birth was not available on the EEG report. Therefore, the total number of Victorian-born infants with epilepsy that is not SEI may be lower than 119. This group mainly contained infants whose epilepsy had (or was expected to have) a ‘benign’ (self-limited) outcome, being those with well-described ‘benign’ epileptic syndromes and other infants with infrequent and/or treatment-responsive seizures (typically of unknown cause). However, this group also contained infants with epilepsies that may or will become ‘severe’ at an older age, including 9 infants with focal epilepsy due to a brain malformation, and 6 with Dravet syndrome. The details of the epileptic syndromes and aetiologies in infants with epilepsy that were ‘not SEI’ are listed below in Table 4.3.

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Table 4.3 Epileptic syndromes in infants with epilepsy not meeting clinical criteria for SEI

Epileptic syndrome

GEFS+/ Dravet syndrome spectrum 15 GGE (other) 9 BFNE/BFNIE/BFIE^12 BFNE/BFNIE/BFIE-like$ 32 SFE 14 Other focal 9 Unknown 28

BFNE = benign familial neonatal seizures, BFNIE = benign familial neonatal-infantile seizures, BFIE = benign familial infantile seizures, FS+ = febrile seizures plus, GEFS+ = genetic epilepsy with febrile seizures plus, GGE = genetic generalised epilepsy, SFE = ‘symptomatic’ focal epilepsy

^Defined as typical clinical features and course with family history of similar, with or without genetic confirmation

$Defined as epilepsy having the typical clinical features and course of BFNE/BFNIE/BFIE, but with no family history of similar

Table 4.4 Aetiologies in infants with epilepsy not meeting clinical criteria for SEI

Cause

Unknown 86* Structural – malformative 12 Structural – acquired 4 Chromosomal – 6 SCN1A – 6# Other genetic 5

*including 1 with Dravet syndrome -like epilepsy and 8 with FS+/GEFS+/ Dravet syndrome spectrum epilepsy who had negative SCN1A testing or who had not been tested.

#5 with Dravet syndrome and 1 with GEFS+

Other groups of infants who may be considered to have a severe epilepsy by other definitions include those with recurrent status epilepticus and those with developmental plateau or regression. It was not possible to accurately determine the number of infants in these groups as this level of detail was not available on most EEG reports and medical records were not reviewed where EEG report detail was sufficient to determine that inclusion criteria had not been satisfied. However, as expected, some infants with

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Dravet syndrome had recurrent status epilepticus and did not meet study criteria for SEI.

4.2 Epidemiology

4.2.1 Incidence of SEI Incidence refers to the number of new cases in an at-risk population in a given time period. For SEI, this at-risk population is all infants live-born in Victoria during 2011- 2013. The live birth rates in Victoria were 71,444 births in 2011, 77,405 in 2012 and 73,969 in 2013, with a total of 222,818 births (www.abs.gov.au). 114 infants with SEI were born in Victoria during 2011-2013. Thus, the crude incidence of SEI in Victoria for infants born during 2011-2013 is 51.2/100,000 live births/year, or approximately 1:2000. The 95% confidence interval for this crude incidence rate is 42.7-61.4/100,000 live births/year5 (Kirkwood, 1988).

The incidence is similar between years if each is calculated individually, each year’s incidence being within the calculated confidence interval – 53.2/100,000/year in 2011 (38 cases), 54.3/100,000/year in 2012 (42 cases) and 45.9/100,000/year in 2013 (34 cases).

A statistical correction for population migration should be applied to the calculated incidence rate to account for the main reason for potentially incomplete ascertainment – namely, that infants with SEI may have moved out of Victoria prior to onset of seizures or prior to presentation to medical care. Using assumptions applied to available data on population migration out of Victoria, an estimated 10,654 infants under two years old born in 2011-2013 moved out of Victoria6 (www.abs.gov.au). Assuming the above

5 Confidence interval calculated according to the formula for ‘Confidence interval for a rate’ from Kirkwood and Steine, where 95% confidence interval = rate/error factor to rate x error factor. The error factor = exp(1.96/√d), where d= the number of events (114 in this study). So, EF = exp(196/√114) = 1.2  EF = 51.2/1.2 to 51.2 x 1.2 = 42.7-61.4/100,000 live births/year. 6 Population migration data from the Australian Bureau of Statistics is reported in 5-year age groups (0-4 years old), without a breakdown of the distribution of each age with in this group. In the five financial years 2010-11 to 2014-5, 43197 0-4 year olds moved interstate or overseas from Victoria (8885 in 2010-

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crude incidence rate, one would expect approximately six infants (5.5) to have SEI in this group. Six more infants would bring the total number to 120, with an adjusted incidence of 53.9/100,000 live births/year, which is well within the calculated confidence interval of the crude incidence rate. As previously noted, two Victorian-born infants with SEI who moved out of the state were actually identified through the ascertainment sources. Thus, one might expect that four children with SEI have not been identified because they moved out of the state.

Given that the crude and corrected incidence rates are so similar, the incidence of SEI will be considered to be the crude incidence, 51.2/100,000 live births/year.

4.2.2 Discussion Comparative incidences and importance of SEI: There is significant variability in the incidence of all infant epilepsy reported in other studies, due to both methods of ascertainment used and to variability in the age range studied. Thus, it is difficult to use most of these studies to estimate the proportion of infant epilepsy that is ‘severe’. Previously reported incidences of all epilepsy in infancy (87-158/100,000 live births/year in the first year of life and 23.4-150/100,000 live births/year in the second year of life) suggest SEI makes up an important proportion, but not necessarily a majority, of infant epilepsies (C. S. Camfield et al., 1996; Casetta et al., 2012; Dura- Trave et al., 2008; Eltze et al., 2013; C. M. Freitag et al., 2001; E. Gaily et al., 2016; Kurtz et al., 1998; Olafsson et al., 2005; Rantala & Ingalsuo, 1999; Wirrell et al., 2011).

2011, 8971 in 2011-2012, 9025 in 2012-2013, 9160 in 2013-2014 and 7156 in 2014-2015). To estimate the total number of infants moving out of Victoria who were under 2 years old (used 2 years rather than 18 months to account for delayed presentations as have done with EEG screening) and born in 2011-2013 from these figures, I have made two assumptions. Firstly, I have assumed equal distribution of ages within this group – that is, 40% of this age group is under 2 years old. Secondly, not all infants under two years old in each financial year would have been born in 2011-2013; the proportion that were has been determined based on the assumption that all infants moved on the last day of the financial year. For the financial year 2010-2011 (moving June 30, 2011), only ¼ infants under 2 years old were born in 2011, thus ¼ of 40% or 10% of the whole 0-4 year old group were assumed to be under 2 years old and born during 2011-2013. Similar calculations applied the following financial years assumes 30% of the group in 2011-2012, 40% in 2012-2013, 30% in 2013-2014 and 10% in 2014-2015. Thus, the estimated number of under two year olds born during 2011-2013 moving out of Victoria is 10,654 (889 in 2010-2011, 2691 in 2011-2012, 3610 in 2012-2013, 2748 in 2013-2014 and 716 in 2014-2015).

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However, a more recent population-based study reported that only 16% of infants 1-24 months old with new onset epilepsy had a ‘more benign evolution’, therefore suggesting the majority of infant epilepsies are severe (Eltze et al., 2013). In our study, approximately half of epilepsies with onset under 18 months old were SEI, the other half being ‘benign’ epilepsies and epilepsies that did not become ‘severe’ until after infancy. This proportion is more in line with the assumptions made from the incidence of all epilepsies, than the data from Eltze et al. The difference in the proportion estimated in this study to that reported by Eltze is likely explained by a difference in the definitions used, namely that epilepsy with ‘more benign evolution’ used by Eltze is not the same as that of epilepsy that is ‘not SEI’ used in this study. The ‘not SEI’ group in this study includes children with epilepsy more severe (either in infancy or after infancy) than in those in their benign group (who presumably had seizure resolution and normal developmental outcome). It is also important to note in this study that infants with epilepsy that is ‘not SEI’ were not studied as comprehensively and systematically as those with SEI. EEG reports and medical records were searched to determine whether or not an infant had SEI, not to definitively establish an epilepsy diagnosis in infants who clearly did not meet the definition for SEI. Therefore, it is possible that some have been erroneously labelled as having epilepsy or not having epilepsy. More detailed study of the group of infants with ‘infant epilepsy – not SEI’ would be required to confidently estimate the proportion of infant epilepsy that is ‘severe’; this is beyond the scope of this study.

While the incidence of infantile spasms and Dravet syndrome have been previously reported, that of other severe infantile epileptic syndromes and SEI as a whole have not. In this study, 74/114 infants had infantile spasms (see Chapter X), which gives an incidence of 33.2/100,000 live births/year (approximately 1:3000). This is similar to previous reports, and therefore provides some independent validation of the ascertainment strategies used in this study (Cowan & Hudson, 1991). The incidence of Dravet syndrome cannot be accurately determined solely from the number of infants with SEI in this study as a number of affected infants did not meet inclusion criteria. However, when infants with Dravet syndrome in both the SEI (4 with Dravet syndrome and 2 Dravet-like) and ‘not SEI’ (5 with Dravet syndrome and 1 Dravet-like) groups are considered, the incidence is approximately 4/100,000 live births/year (approximately

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1:25,000) for infants with Dravet syndrome and 5.4/100,000 live births/year (approximately 1:19,000) for infants with both Dravet and Dravet-like syndromes. Although these figures are less robust than the other incidence figures presented in this chapter due to less rigorous study of those not meeting inclusion criteria, they are nevertheless comparable to previous studies (Bayat et al., 2015; Wu et al., 2015).

External validity: It is important to consider whether the incidence of SEI in Victoria is likely to be representative of the incidence in other populations. It is likely that the rates of genetic SEI are similar across populations given that mutations in a large number of genes can cause SEI and each is thought to be collectively uncommon (rev in (McTague et al., 2016)), thereby reducing the probability of variability in SEI incidence due to variability in incidence of a single genetic cause between populations of different ethnic background. The rates of acquired causes of SEI are likely to vary across populations though, being higher in countries with poorer access to medical care, and higher rates of central nervous system infections and perinatal medical problems such as HIE. Therefore, the overall rate of SEI in countries with equivalent quality of, and access to, medical care as in Victoria is likely to be similar to that calculated here. On the other hand, the rate of SEI in countries with limited access to health care would be expected to be higher.

4.3 Conclusions

This is the first population-based study of the incidence of all severe epilepsies in infancy. The high quality data obtained in this study shows that SEI are relatively common for such a severe group of disorders. SEI has a higher incidence than some well-known severe neurologic disorders of infancy and childhood, including neurofibromatosis type 1 (1:2700), Duchenne muscular dystrophy (1:4000) and spinal muscular atrophy (1:12,000) (Arkblad, Tulinius, Kroksmark, Henricsson, & Darin, 2009; Evans et al., 2010; Mendell et al., 2012). Although comparison with the incidence of these single disorders is perhaps unfair, the comparison does highlight that SEI is a relatively frequent group of severe seizure disorders that pose a significant health burden for the community.

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Subsequent chapters of this thesis will examine the incidence of each epileptic syndrome, and of the different aetiologies, including genetic causes of SEI, data that is critical to guiding rational use of diagnostic investigations and informing priorities for translational research.

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Chapter 5: Aetiologies

5.1 Aetiologies

5.1.1 Individual aetiologies The aetiology of SEI was determined in 76 (67%) infants. These infants had 34 different aetiologies for SEI. FCD was the most common cause, identified in 14 (12%) infants. Trisomy 21 and TS were each diagnosed in 5 (4%) of patients. Table 5.1 lists the aetiologies ranked by frequency. The aetiology is unknown in 38 (33%) infants. The aetiologies in individual patients, including mutation details where relevant are listed in Appendix I.

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Table 5.1 Aetiologies of SEI in 114 infants

Aetiology N

Focal cortical dysplasia 14 Trisomy 21 5 Tuberous sclerosis 5 Perinatal hypoxic ischaemic encephalopathy 4* KCNQ2 mutation 3 Malformation of cortical development (other) 3 Mitochondrial disorder 3 Periventricular leukomalacia 3 Polymicrogyria 3 SCN1A mutation 3 Complicated meningitis 2 Lissencephaly 2 Perinatal HIE and hypoglycaemia 2 Perinatal stroke 2 SCN2A mutation 2 SCN8A mutation 2 Achondroplasia 1 Aicardi syndrome 1 Aicardi-Goutières syndrome 1 Chromosome 2q24.3 deletion (incl. SCN1A and SCN2A genes) 1 Chromosome 15q21.3q22.2 deletion 1 Isodicentric chromosome 15 1 KCNT1 mutation 1 Molybdenum cofactor deficiency 1 Perinatal/neonatal ischaemic injury (mechanism unknown) 1 PNPO deficiency 1 Pontocerebellar hypoplasia 1 SMC1A mutation 1 Sotos syndrome 1 Sturge-Weber syndrome 1 SYNGAP1 mutation 1 Tay-Sachs disease 1 TBC1D24 mutation 1 Wolf-Hirschhorn syndrome 1 Unknown 38

Total 114

*1 infant with possible underlying genetic aetiology for HIE due to additional clinical features including congenital cataracts, which are not accounted for by HIE (suspected genes COL4A1 or COL4A2 (Meuwissen et al., 2015)), awaiting WES

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5.1.2 Aetiologic groups Fourteen infants (12%) had an acquired aetiology, such as hypoxic-ischaemic encephalopathy, periventricular leukomalacia and perinatal stroke, in which genetic factors were not considered primary. The remaining 100 (88%) had genetic or unknown aetiologies.

Using the ILAE 2010 classification, structural causes were most common aetiologic group. Structural causes were seen in 45 (39%) infants, metabolic disorders in 6 (5%) and ‘genetic causes’ in 26 (23%). Classifying aetiologies according to the timing of (aetiologic) onset, 62 (54%) had aetiologies of prenatal onset, 13 (11%) perinatal onset and 1 (1%) postnatal onset.

In this chapter, the term ‘genetic’ has two meanings. Firstly, it is part of the ILAE 2010 classification of aetiologic groups, used to refer to non-malformative, non-metabolic conditions with a genetic basis (Berg et al., 2010). Secondly, given metabolic and many structural conditions have a genetic basis, it is also used to refer to any structural or functional brain disorder due to a genetic aberration. It was helpful for clarity to distinguish the two uses of this term; thus ‘genetic’ (in inverted commas) denotes the ILAE 2010 classification group, and genetic (no inverted commas) will be used to mean any condition with a genetic basis, including malformative and metabolic aetiologies.

Two minor modifications to the ILAE 2010 classification were utilized. Firstly, genetic and acquired conditions will be separated given the differences in mechanisms and implications of such diagnoses. Secondly, the ‘genetic’ group will be separated into two groups, chromosomal and single gene disorders. Thus, five groups were used

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Table 5.2 Aetiologies of SEI using a modified classification

Aetiologic category N Aetiologies

Perinatal HIE 4, Periventricular leukomalacia 3, complicated meningitis 2, Perinatal HIE and hypoglycaemia 2, perinatal 14* Acquired conditions stroke 2, perinatal/neonatal ischaemic injury (mechanism unknown) 1 Focal cortical dysplasia 14, tuberous sclerosis 5, polymicrogyria 3, malformation of cortical development (other) 3, lissencephaly Brain malformations 31 2, malformation of cortical development (other) 2, achondroplasia 1, Aicardi syndrome 1, pontocerebellar hypoplasia 1, Sturge-Weber syndrome 1 Mitochondrial 3, molybdenum cofactor deficiency 1, PNPO 6 Inborn errors of metabolism deficiency 1, Tay-Sachs disease 1 Trisomy 21 5, chromosome 2q24.3 deletion, Wolf-Hirschhorn Chromosomal abnormalities 9 syndrome, isodicentric chromosome 15, chromosome 15q21.3q22.2 deletion SCN1A mutation 3, KCNQ2 mutation 3, SCN2A mutation 2, SCN8A mutation 2, Aicardi-Goutières syndrome 1, KCNT1 Neuronal excitability 16 disorders mutation 1, SMC1A mutation 1, Sotos syndrome 1, SYNGAP1 mutation 1, TBC1D24 mutation 1

Unknown ^ 39

Total 114

*1 infant with possible underlying genetic aetiology for HIE (suspected genes COL4A1 or COL4A2), awaiting WES

^See table 5.4 for suspected causes in infants with unknown aetiology

Acquired conditions: Acquired conditions occurred in 14 (12%) infants, and made up one-third of all structural aetiologies. They occurred in the perinatal period or first week of life in all but one infant. This infant, born at 31 weeks gestation, had complicated meningitis at 3 months of age, this being within the ‘neonatal’ period for corrected gestational age (43 weeks).

Brain malformations: Malformative conditions were the most common aetiologic group, occurring in 31 (27%). All but one malformation primarily or solely involved the cortex. One infant had a malformation which is considered primarily a hindbrain malformation (pontocerebellar hypoplasia). Malformations of cortical development were most commonly focal (e.g. FCDs) and multifocal (e.g. TS), but some diffuse malformations (e.g. lissencephaly) were also seen (Figure 5.1).

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Figure 5.1 Examples of malformations of cortical development in infants in this study.

A) Coronal T2-weighted imaging in infant IEE14001 showing a focal cortical dysplasia in the right frontal lobe (arrow)

B) Coronal T1-weighted imaging in infant IEE13038 showing features of tuberous sclerosis including tubers in the left and right frontal, and left temporal lobes (arrows)

C) Axial T2-weighted imaging in infant IEE13045 showing posterior-predominant lissencephaly with a cell-sparse zone (arrow), typical of that seen with LIS1 mutations

Metabolic disorders: Mitochondrial conditions were the most common metabolic disorder, diagnosed in three infants. Three other disorders, involving three different metabolic pathways, were seen in just one infant each.

Chromosomal abnormalities: Nine (8%) infants had a chromosomal abnormality, making up approximately one-third of those with an identified ‘genetic’ aetiology. Trisomy 21 was the most common chromosomal abnormality, in 5/9 infants. The remaining infants had chromosomal variants that have been previously associated with seizures, being well-described conditions in three (Arya, Kabra, & Gulati, 2011; A. Battaglia, Filippi, South, & Carey, 2009; Finucane et al., 1993; Joshi et al., 2016) and reported in a small number of cases in one (15q21.3q22.2 deletion) (Lalani, Sahoo, Sanders, Peters, & Bejjani, 2006; T. Yamamoto et al., 2014). All chromosomal anomalies occurred de novo in the infant.

Single gene disorders: Sixteen (14%) had a single gene disorder. Mutations in ten individual genes involved in a number of different aspects of neuronal function were identified. Mutations in genes responsible for ion channel function (‘channelopathies’) were the most common group of disorders, seen in 11 (69%) infants with mutations of

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sodium channel genes in seven and potassium channel genes in four. Three infants had mutations affecting the regulation of DNA transcription and/or RNA translation (NSD1, SMC1A, RNASEH2B) and two infants had mutation in genes affecting synaptic function (SYNGAP1, TBC1D24). Thirteen infants had autosomal dominant disorders. Mutations arose de novo in 7/13, were inherited from a parent with somatic mosaicism in 2/13, occurred in the context of presumed parental gonadal mosaicism in 1/13 (affected sister, no parental somatic mosaicism detected), and were of unknown inheritance in 3/13. Two infants had autosomal recessive disorders; parents were heterozygous in both instances. One infant had an X-linked disorder7.

5.1.3 Genetic basis of malformative and metabolic conditions

A genetic basis was sought in all infants with a metabolic disorder and 19/31 infants with brain malformations (Table 5.3). 5/6 (83%) infants with metabolic disorders had the causative (or potentially pathogenic in the infant with a novel homozygous missense mutation in FARS2).8 The remaining infant had a familial mitochondrial disorder, with complex IV deficiency proven in a sibling. WES of this infant, two affected siblings, and other unaffected family members, did not reveal a cause. A genetic basis was confirmed in 9/19 infants with brain malformations who underwent testing. The genetic basis is presumed in 3/12 infants with malformations who did not have genetic testing; these infants having clinical features highly associated with one or two genes (posterior-predominant lissencephaly presumed associated with a LIS1 mutation, Sturge-Weber syndrome presumed associated with a mosaic GNAQ mutation and TS presumed associated with a TSC1 or TSC2 mutation).

7 The infants in whom inheritance of the variant is pending (3) or not tested (1) are one infant with a frameshift mutation in SMC1A which fits her phenotype, and three infants with missense mutations that are predicted damaging in KCNQ2. The phenotype in these three infants is consistent with KCNQ2; it was the top-ranked gene (following my clinical assessment) in 2/3 infants. One mutation (in the infant whose parents were not tested) has been previously reported pathogenic. 8 The FARS2 gene fits the infant’s epilepsy, developmental, biochemical and imaging phenotype well, but does not explain some other clinical features such as an encephalocoele and small kidneys. It is suspected that this infant has two Mendelian disorders).

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Table 5.3 Genetic basis of malformative and metabolic aetiologies

Pathogenic/potentially pathogenic genetic basis No confirmed genetic basis identified

Aetiology N Testing negative, No testing N % Genes N identified a done VOUS, or pending

Structural-malformative 5 (3 WES negative, 1 WES VOUS Focal cortical BRAF (1), DEPDC5 in MTOR 14 3 21 11 6 dysplasia (1), NPRL3 (1) gene, 1 TSC1/2 sequencing negative) Tuberous 1 (presumed 5 4 80 TSC1 (1), TSC2 () 1 0 sclerosis TSC1/2) 1 (WES on Polymicrogyria 3 0 0 N/A 3 2 brain tissue) 1 (presumed Lissencephaly 2 1 50 LIS1 (1) 1 0 LIS1) Malformation of 1 (WES, cortical 3 0 0 N/A 3 1 result development pending) (other) 1 (WES Aicardi syndrome 1 0 0 N/A 1 0 negative)

Achondroplasia 1 1 100 FGFR3 0 N/A N/A

1 (TSEN54 Pontocerebellar 1 0 0 N/A 1 0 sequencing hypoplasia negative) Sturge-Weber 1 (presumed 1 0 0 N/A 1 0 syndrome GNAQ)

Metabolic Mitochondrial FARS2 (1), 1 (WES 3 2 67 1 0 disorder NDUFAF6 (1) negative) Molybdenum 1 1 100 MOCS2 (1) 0 N/A N/A cofactor deficiency

PNPO deficiency 1 1 100 PNPO (1) 0 N/A N/A

Tay-Sachs 1 1 100 HEXA (1) 0 N/A N/A disease

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5.1.4 Genetic aetiologies in infants with similarly affected siblings Seven infants from six families had one or more similarly affected siblings. In just one family did both affected siblings meet inclusion criteria for this study; in the remaining families the older sibling was born prior to the study period. Two families had additional affected members, being first cousins in one, and second and third degree relatives in multiple branches of the family in the other. At least one affected family member in four families was deceased. The genetic basis was investigated in all six families, and identified in four. Two families had autosomal recessive conditions. Two had autosomal dominant disorders, one involving a gene known to have variable penetrance and severity inherited from an unaffected parent, and the other presumed to be due to parental gonadal mosaicism. The remaining two families, who are presumed to have autosomal recessive conditions, had negative WES performed on at least two affected individuals (WES gene panel in one family, unbiased WES in the other).

5.1.5 Unknown aetiologies 38 infants have an unknown aetiology for their SEI. Eight have a clinically suspected cause that has not been confirmed (Table 5.4). Six infants are thought likely to have an occult FCD; these infants were not further investigated as seizures have ceased. A single gene disorder is thought likely in two patients.

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Table 5.4 Suspected aetiologies in infants with unknown cause for SEI

Infant Suspected aetiology Evidence

IEE11009 Focal cortical dysplasia Unifocal seizures prior to spasm onset (bilateral asymmetric tonic  right leg clonic), possible left cingulate dysplasia on MRI.

IEE12003 Focal cortical dysplasia Prominent left arm paresis at the age at which spasms were present, no definite dysplasia on MRI.

IEE13003 Focal cortical dysplasia Persistent right frontal interictal epileptiform activity and probable focal slowing (EEG never normalised), area of abnormal sulcation in right frontal lobe on MRI without definite dysplasia.

IEE13036 Focal cortical dysplasia Continuous right centroparietal interictal epileptiform discharges, asymmetric spasms, right arm paresis at the age at which spasms were present, no definite dysplasia on MRI.

IEE13047 Focal cortical dysplasia Unifocal seizures prior to spasm onset, which were ongoing during time having spasms, continuous left centroparietal interictal epileptiform activity, and focal seizures recorded, no definite dysplasia on MRI.

IEE14023 Focal cortical dysplasia Persistent right mid-posterior temporal interictal epileptiform activity and focal slowing (EEG never normalised), area of abnormal sulcation in right occipital lobe on MRI without definite dysplasia.

IEE12038 SCN1A mutation Dravet syndrome clinically, no genetic testing performed.

IEE14054 AARS mutations (biallelic) Additional clinical features of axonal neuropathy, congenital vertical tali and marked growth restriction. WES gene panel result pending.

Thirteen infants had one or more variants of unknown significance identified on WES gene panel. Additional information such as functional studies of the variant or additional reports of similarly affected individuals will be required to determine whether a variant is the cause of an infant’s SEI. The results of the WES gene panel are pending in five infants.

17 infants had no cause identified on research genetic testing. 5 infants with unknown aetiology following clinical investigation and research imaging review did not consent to this study, and did not have any genetic testing apart from chromosomal microarray.

5.1.6 Commentary This study has identified an aetiology for SEI in two-thirds of infants. Over 200 aetiologies for infant-onset seizures are reported; 34 were identified here. Like in previous studies, a small number of aetiologies, such as TS and (/or) trisomy 21, were

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seen relatively commonly (each in 4% in this study) (Chevrie & Aicardi, 1977; Czochanska et al., 1994; Eltze et al., 2013; E. Gaily et al., 2016; Matsumoto et al., 1983; Osborne et al., 2010), and most of the remaining aetiologies were individually uncommon . Some well-established causes of infantile epilepsy, such as CDKL5 encephalopathy and STXBP1 encephalopathy were not seen at all. This is perhaps not surprising given the size of the study and previous studies that suggest that even the most common genetic (non-structural, non-metabolic, non-chromosomal) causes of severe epilepsy (other than SCN1A) each account for only 1-2% of the whole group (Carvill, Heavin, et al., 2013).

In this study, the most common group of conditions was brain malformations, and the most common individual cause was FCD, diagnosed in 12% and suspected in a further 5%. The number of infants with FCD in this study is considerably higher than that reported in three recent studies of infant epilepsies; FCD was reported in 3% of all first- year of life onset epilepsies, 4% of all epilepsies with onset between 1-24 months old (both studies population-based) and 0.5% of infants with infantile spasms (not population-based) (Eltze et al., 2013; E. Gaily et al., 2016; Osborne et al., 2010). The reasons for this discrepancy may be partly related to the patient cohort, as one would perhaps expect a lower proportion with FCDs among a group of all infant epilepsies than a group with SEI. The quality of brain imaging is likely also relevant; older studies that relied on CT scans or lower field strength MRI would not be expected to resolve small FCDs. It is likely though, that FCD is still under-recognised. Certainly, in this study, previously unrecognized FCDs were identified on detailed review of brain imaging or repeat imaging. That possibility is also supported by earlier studies of PET in infants and children with refractory spasms of unknown cause, which showed uni- or multifocal abnormalities in most infants (unifocal in approximately 30%), which were presumed to represent dysplastic lesions (Chugani & Conti, 1996). In the absence of an alternative diagnosis, clinicians should have a high level of suspicion of an FCD, particularly where seizures are refractory, as epilepsy surgery may be curative. Given the treatment implications, prospective studies of brain imaging are warranted to investigate whether more infants with an unknown aetiology harbour occult FCDs, and to determine whether there is additional diagnostic yield from very high quality brain

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imaging (e.g. using combined techniques such as 3T MRI-PET scans) over current standard 1.5 or 3T MRI.

Changes in aetiologic classifications have occurred over time, as understanding of the aetiologies of epilepsy advances, making comparisons between newer and older studies difficult. The earliest studies classified aetiologies by timing of onset. The proportion of infants with postnatal onset of the aetiology (e.g. meningitis in infancy) is similar in this study to previous (1% here vs 1-4% in other studies). Here, perinatal aetiologies are about half as common as previously reported (11% vs 20-23%), which may reflect improvement in perinatal medical care. It is possible also that there is now a reduced tendency to assume a perinatal aetiology unless there is evidence of major medical issues in that period. The most striking difference though is in the proportion of infants with aetiologies of prenatal onset – i.e. genetic and presumed genetic conditions. In earlier studies, these were seen in 18-20%. In this study, the majority of aetiologies were of confirmed (54%) and presumed (34%, i.e. the group of infants with unknown aetiology in whom there was no evidence of a peri- or postnatal insult) prenatal onset (Chevrie & Aicardi, 1977; Czochanska et al., 1994; Matsumoto et al., 1983). Advances in brain imaging and genetic diagnostic techniques likely account for this difference. Between this classification and the current one came a classification that groups aetiologies as symptomatic, idiopathic and cryptogenic. This terminology was used so variably in the literature as to make comparisons with this study difficult even before one considers advances in diagnostic techniques. Thus, that classification has not been further considered here.

Using the ILAE 2010 classification of structural vs metabolic vs ‘genetic’ causes, structural aetiologies were the most common group, diagnosed in 39%, and metabolic disorders were identified in only 5%. Both figures are similar to recent studies. 22% of infants in our study had a ‘genetic’ aetiology, slightly higher than the 17% reported by Gaily et al and significantly higher than studies by Eltze et al and Osborne et al (Eltze et al., 2013; E. Gaily et al., 2016; Osborne et al., 2010). The only ‘genetic’ diagnoses made in the latter two studies were chromosomal disorders, and two SCN1A mutations (in 57 infants in the study by Eltze et al), as access to other genetic testing was not available. The Gaily study included five familial cases (unclear if these were sib pairs,

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parent-child pairs or more distant relatives) with no identified gene, making up approximately two-fifths of their ‘genetic’ group. In my study, such cases have been classified as unknown rather than genetic. Therefore, 11-17% of the infants in the Gaily study had an identified ‘genetic’ cause (depending on the number of sib pairs with familial epilepsies), including a number of infants with chromosomal disorders and single gene mutations that were identified on single gene testing targeted to the epilepsy phenotype (SCN1A, CDKL5, SCN2A etc.). That study did not have access to NGS. Thus, the differences in the proportion of infants with a ‘genetic’ aetiology across studies is due in large part to the genetic testing performed, with additional contributions from differences in the patient populations and inclusion (or otherwise) of familial cases without an identified gene in the ‘genetic’ group.

A limitation and criticism of the current aetiologic classification is that it doesn’t clearly convey that many structural conditions and all metabolic disorders have a genetic basis. It is useful at a clinical level to consider which aetiologies are acquired (in which genetic factors may play a part but are not considered primary) and which are genetic. Therefore, for this study, a modified classification was used, classifying aetiologies as acquired, genetic or unknown, with the genetic group having subgroups for brain malformations, inborn errors of metabolisms, chromosomal abnormalities and single gene disorders. The unknown group was deemed to have a presumed genetic cause because of an absence of evidence for an acquired aetiology, although clear supportive evidence such as a family history of similar conditions was absent in most. The vast majority of infants had a genetic or presumed genetic basis for SEI, with acquired aetiologies identified in just 12%. Among those with genetic/presumed genetic aetiologies, brain malformations were seen in 31% of infants and single gene disorders in 16%. These two groups of disorders should be seen as the main causes of SEI.

The proportion of infants with acquired aetiologies in this study is slightly lower than (but not significantly different from) recent studies of all infant epilepsies (12% compared with 15% (χ2 = 0.29, df=1, p=0.59) and 16% (χ2 = 4, df=1, p=0.53)), and approximately half that seen in UKISS (23%, χ2=5.6, df=1, p=0.02) (Osborne et al., 2010). Given that infants with acquired aetiologies most commonly present with spasms, a higher rate in that study is not unexpected; in my study, acquired aetiologies

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were seen in 16% of infants with spasms. This study, and the three previous studies were all conducted in countries with high quality antenatal and perinatal care in which the rate of major perinatal complications is low. The proportion of infants with SEI due to acquired injuries would be expected to be higher in countries with higher rates of perinatal morbidity.

Brain malformations were focal or multifocal in approximately three-quarters (24/31) of malformations, or approximately one in five of all infants with SEI, and diffuse in one- quarter. The focal and multifocal groups are made up largely of infants with FCD, focal areas of polymicrogyria, and TS, all of which may be amenable to curative epilepsy surgery. At RCH, which has a large epilepsy surgery program, fourteen infants had had epilepsy surgery at the time of writing, but only five prior to two years old (further discussed in the Clinical Features chapter). While the overall proportion of infants with malformations having epilepsy surgery for refractory epilepsy at our centre is high, there is still work to be done to improve the number of infants having surgery in early life, at which age the developmental benefits of stopping seizures are greatest (Loddenkemper et al., 2007).

Metabolic aetiologies were not common, seen in this study in 5% of infants. It is unlikely that many (if any) metabolic diagnoses have been missed, as most infants with unknown cause and refractory seizures had extensive investigation for these. Other studies have seen similarly low rates, which should prompt a shift in previously-held ideas that metabolic disorders are a common cause of SEI.

Single gene disorders were identified in a larger proportion of infants in this study than in previous reports, which reflects availability of genetic testing. While a large variety of single gene disorders, involving different cellular pathways and mechanisms, are recognized and, indeed, were diagnosed here, it is striking in the current study that channelopathies made up more than half of all of this group, and 10% of the whole SEI population. While channelopathies have long been recognized as the major cause(s) of ‘benign’ familial neonatal and infantile epilepsies (Grinton et al., 2015; Zara et al., 2013), their prominence (as a proportion of all single gene disorders) had not quantified in previous studies of NGS in severe epilepsies as the patient cohorts in those studies

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were not population-based and typically tested a broader age range. This finding is important, as it may suggest common mechanisms for early life severe epilepsies, with potential implications for development of novel therapies for SEI.

In this study, an underlying genetic basis was identified in most infants with a metabolic disorder, and almost half of those with a brain malformation who underwent genetic testing. Twelve infants with brain malformations did not undergo any genetic testing, and two with negative testing only had one or two genes tested. Thus, the proportion of this group with genetic cause identifiable on testing of non-brain DNA is not yet known; this bears further study. A number of infants who have not been tested have conditions in which the genetic basis is well-established (e.g. posterior-predominant lissencephaly associated with LIS1 mutations, and TS), and would be expected to have a positive genetic diagnosis made if testing were done. It is likely though that not all infants with malformations will have a genetic cause identified. Some conditions, such as Aicardi syndrome, are presumed to have a genetic basis but the causative gene(s) is not yet known. Some malformations, such as polymicrogyria, are not always genetic, being seen in some infants with insults acquired in early gestation such as CMV infection and vascular injuries (rev in (Stutterd & Leventer, 2014). Finally, not all malformations will have a genetic mutation in the germline (nor somatic mosaic in multiple tissues), some being due to somatic mutations confined to brain tissue. For obvious reasons, these are difficult to identify, although perhaps easier in this patient group than in patients with malformations without epilepsy, as some will undergo epilepsy surgery at which time brain tissue can be studied for mosaic mutations. Mosaic mutations in the brain are presumed to underlie many focal malformations such as FCD. In recent years though, germline mutations have been identified in patients with FCD, mainly in mTOR pathway genes including DEPDC5, MTOR, NPRL2 and NPRL3 (Dibbens et al., 2013; Moller et al., 2016; Ricos et al., 2016; Scerri et al., 2015; Sim et al., 2016), dominantly inherited genes which display variable penetrance and a spectrum of phenotypic severity. Prior to this discovery, cases of familial FCD were very rare, and the recurrence risk of FCD very low. In this study, 3/14 infants with FCD had a germline mutation identified (NPRL39, DEPDC5 and BRAF), one of which was

9 Identified via another study, published in Annals of Neurology (Sim et al, 2016)

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inherited. It is possible that the recurrence risk is higher than previously appreciated; multigene testing in the infants with FCD not yet tested would be useful in further investigating this possibility.

Mutations in autosomal dominant and recessive, X-linked and mitochondrial genes are reported in SEI. De novo dominant mutations have emerged in recent years as a major cause of SEI, which is not surprising as most infants with SEI have unaffected parents and many with SEI would not be expected to reproduce and pass on a mutation if they survive to reproductive age (rev in (McTague et al., 2016). In this study, de novo dominant mutations were the most commonly seen, but autosomal recessive, X-linked and presumed parental gonadal mosaicism were also seen. Thus, in some infants with SEI there is a significant risk of the parents having a second affected child, as evidenced by six families in this study having two affected children. Those with metabolic disorders are at particularly high risk, as most of these disorders have autosomal recessive inheritance. The risk of having a second child affected by such a severe disorder is a strong argument for early genetic testing and genetic counselling.

One third of infants in this study remain without a diagnosis. This compares favourably to previous studies of all epilepsies in infants, in which approximately half had no identified cause (Eltze et al., 2013; E. Gaily et al., 2016). The difference in yield in this study relates to research imaging and genetic testing and is discussed in the Diagnostic Investigation chapter. While those with unknown cause have recently been conceptualized as having single gene disorders, some infants in this study with previously unknown cause had an FCD identified through research imaging review and FCDs are strongly suspected in a number of infants in whom the cause remains unknown after research imaging and genetic testing. Thus, this study suggests that the group with unknown aetiologies is includes infants with single gene disorders and occult malformative causes.

The relationships between aetiology and the clinical features, in particular epilepsy phenotype, development and survival, are discussed in the Clinical Features chapter. The Diagnostic Investigation chapter will discuss the use of diagnostic investigations in SEI, including how and when aetiologic diagnoses were made.

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5.2 Incidence of aetiologies

The incidences of particular groups of SEI aetiologies are listed in Table 5.5, by known or unknown cause and acquired vs genetic/presumed genetic aetiology. The incidence of any structural (encephaloclastic or malformative) aetiology is 19.3/100,000 live births/year. Malformations of cortical development (all but one malformative cause) occurred in 12.6/100,000 live births/year.

Table 5.5 Incidence of SEI by aetiologic groups using a modified classification

Incidence 95% confidence interval Cause N (/100,000 live births/year) (/100,000 live births/year) Unknown 38 17.1 15.7-18.5

Acquired 14 6.3 4.6-8.0

Brain malformations 31 13.9 12.5-15.3

Inborn errors of 6 2.7 0.5-4.9 metabolism Chromosomal 9 4.0 2.1-5.9 abnormalities

Single gene disorders 16 7.2 5.6-8.8

Total 114 51.2 42.7-61.4

The incidence of SEI due to focal cortical dysplasia (N=14), the most common aetiology, is at least 6.3/100,000 live births/year. The incidence of SEI due to channelopathies (N=12, 11 with single gene basis and 1 with a chromosomal abnormality affecting ion channel genes) is at least 5.4/100,000 live births/year. The incidence of SEI due to trisomy 21 and tuberous sclerosis (each N=5) is 2.3/100,000 live births/year for each condition.

5.2.1 Commentary This population-based study has provided incidence data for aetiologic groups and some individual aetiologies of SEI. The incidence of brain malformations and single gene disorders should be considered structural-malformative minimum estimates given these aetiologies are also presumed to be present in some of the infants with SEI of unknown cause.

The incidence of SEI aetiologies is less clinically useful than the proportion of infants with particular aetiologies and aetiologic groups among the whole SEI population; this is discussed in the Aetiologies section above. These data do, however, allow estimates

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of the proportion of infants with a particular aetiology who develop SEI. Prior studies have looked at the proportion who develop infantile spasms among large groups of infants with trisomy 21 (2.6%) and HIE (5%) (Goldberg-Stern et al., 2001; Inoue et al., 2014). The figures calculated in this study of SEI are similar10, which is not surprising given most infants with epilepsy due to these aetiologies present with spasms (Watanabe et al., 1980), and provide further validation of the study methodology.

10 Trisomy 21 population incidence is 1:1000-1100 live births (ref: www.who.int/genomics/public/geneticdiseses/en/index1.html). The incidence of T21-SEI in this study is 2.3/100,000/year or ~1:43,000. Combining these figures suggests that approximately 1:40 (1:39 if incidence 1:1100, 1:43 if incidence 1:1000) infants, or 2.5%, with T21 will develop SEI. The incidence of moderate-severe HIE in developed countries is approximately 1:1000-2000 live births. The incidence of HIE-SEI in this study is 2.3/100,000/year or ~1:43,000. Combining these figures suggests that 1:22-1:43 infants (2.3-4.5%) with moderate-severe HIE will develop SEI.

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Chapter 6: Clinical features

Medical record review was performed in all 114 infants. 58 were assessed clinically by me at 0.2-4.2 years old (median 1.9 years, interquartile range (IQR) 1.5 years).

At the time of writing, it was 2.6-5.6 years since the infants’ dates of birth (surviving infants 2.7-5.6 years). As the youngest surviving infants are not yet three years old, in order to standardize reporting, clinical features are reported to two years old unless otherwise stated. Follow-up was available to two years old in all but four infants who were lost to follow-up. These infants were 11-21 months old at the date of the most recently available medical records11. For these infants, the information available in the most recent medical records was used when reporting seizure and developmental outcomes.

6.1 Demographics

61 (54%) infants were male and 53 (46%) female. 18 (16%) were deceased by two years old (further discussed in Survival section below). A family history of epilepsy or FS in a first or second degree relative was present for 22 (19%) infants, absent for 79 (69%) and unknown for 13 (11%). Seven (6%) infants had one or more similarly affected sibling(s). Eight (7%) infants were the product of a consanguineous union.

6.2 Epilepsy

6.2.1 Commentary on methodology Prior to reporting on electroclinical phenotypes, it is useful to consider how seizure types and epileptic syndromes were applied in this study as this influences the reading

11 These infants were: two with trisomy 21 no longer seen at RCH. Both had developmental delay but no seizures at last follow up (11 and 17 months old). One with a perinatal stroke moved overseas. At the time of last follow-up (21 months), this infant had global developmental delay (not walking, mild language delay). One with unknown aetiology, and global developmental delay but no ongoing seizures, was lost to follow up at 11 months old. This infant’s treating clinician had advised that his family should not be approached to be involved in this study due to major social concerns.

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of this chapter, pre-empting some of the concerns and questions the reader may have, and explaining the reasons for making particular choices in the methods of classification.

The electroclinical phenotypes in infants with SEI have been studied systematically and comprehensively here. A wealth of clinical information (from medical records +/- clinical review) and interictal EEG was available for all infants, and ictal EEG and video and/or home video of seizures were available in most. Seizure semiology, videos, interictal and ictal EEG recordings were reviewed by two epileptologists (Katherine Howell and Simon Harvey) to determine the electroclinical phenotype. The approach of systematically reviewing all the available primary data on all infants is an advantage of this study as it enables a more detailed determination of the accuracy of reported seizures types and epileptic syndromes, and greater consistency in the findings reported. Some (Eltze et al., 2013), but not all (E. Gaily et al., 2016), previous population-based studies of the electroclinical phenotypes in all infant epilepsies have used this approach. Compared to prior studies, this study is well-placed to describe the whole spectrum of ‘severe’ infant epilepsies, as all but one previous study of all epilepsies did not include infants with epilepsy onset in the first month of life, and the study that did include neonates did not report on epilepsy evolution or seizure outcomes (E. Gaily et al., 2016).

Despite the detailed information available and the expertise of the clinicians, classification of seizures and epileptic syndromes was difficult in many patients. As has been previously reported, it can be difficult in infants to determine whether a seizure is focal or generalized based on semiology alone (M. S. Duchowny, 1987; Hamer et al., 1999; Nordli et al., 1997). Examples of this include seizures with symmetric or asymmetric tonic semiology that may have focal or generalized ictal rhythms, and seizures with hypomotor features, which may have a focal basis or represent a subtle generalized tonic seizure. Thus, there is a higher reliance on additional information such as ictal EEG recordings to accurately classify seizures. As ictal EEG recordings were not available in all infants. It is possible, or even likely, that some have been misclassified.

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Seizure types were classified according to the 2016 ILAE classification, with two modifications to simplify use (http://www.ilae.org/visitors/centre/Class-Seizure.cfm). Firstly, epileptic spasms were considered a single category (not divided into focal/generalized/unknown). Focal seizures were divided only into motor and non- motor subgroups, without further subgrouping by factors such as awareness, given this can be difficult to determine in infants. Data on classification of seizures in infants in this study according to the 1981, 2010 and 2016 versions of the ILAE seizure type classification, and a discussion of their ease of use in this age group is provided in Appendix J.

It is well-recognised that the electroclinical phenotype in many infants is not typical of one of the well-described ILAE epileptic syndromes, some estimating that ‘complex’ phenotypes are seen in half of infants with epilepsy (Eltze et al., 2013). The term ‘early- onset epileptic encephalopathy’ has emerged to describe this complex group (for those with onset under 3 months old), but its use is very broad and therefore this group includes infants with quite disparate phenotypes (Allen et al., 2016; Mastrangelo, 2015; Ohba et al., 2014). The well-described epileptic syndromes have been extremely useful clinically, having implications for treatment and outcomes, as well as pointing to possible or likely underlying aetiologies (rev in (Bureau et al.; McTague et al., 2016)).

Thus, I felt it important to attempt to subclassify the group of infants with ‘complex’ phenotypes, in case grouping previously unclassified infants was similarly clinically useful. The approach used was to determine which ILAE syndrome the infant’s epilepsy most closely matched. Categories ‘[ILAE syndrome]-like’ and ‘[ILAE syndrome]-plus were applied if an electroclinical feature was missing or different from the prototypic ILAE syndrome (-like) or an additional feature was present (-plus). Similar approaches have been used previously, such as the use of the term ‘Dravet-syndrome-like’ to describe epilepsies (e.g. PCDH19-associated epilepsy) with similar features to prototypic Dravet syndrome). In some cases, such as this one, an initial categorisation as a ‘variant’ of an established epileptic syndrome led to further phenotypic studies that have demonstrated that the ‘variant’ is in fact a recognizable epileptic phenotype clearly distinct from the prototypic syndrome (Depienne, Bouteiller, et al., 2009; Trivisano et

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al., 2016). Other focal and generalized and mixed categories were used where the –like and –plus syndromes were not appropriate.

Using this system, we were able to allocate a category to all infants. Given the detailed electroclinical information available on most infants, we were confident in most cases that the syndromes were used as they were defined. For a few infants with focal or multifocal epilepsies, it was not clear if there was one or more seizure types and therefore difficulty in placing them in a ‘unifocal’ or ‘multifocal’ category. The additional group ‘focal – other’ was used for these infants, but may have been able to be discarded if more ictal recordings were available. Following our review, our designated epileptic syndromes were reviewed with my co-supervisor, Prof Ingrid Scheffer. Overall, it was decided that, for some aspects of this study, it would be reasonable to ‘collapse’ the prototypic and variant syndromes in a single group (e.g. ‘EIEE and variants’ rather than the three groups ‘EIEE’, ‘EIEE-like’ and ‘EIEE-plus’). There were a number of reasons for this. In some cases, we had applied a stricter definition of the prototypic syndrome than is commonly used. An example of this is EIEE, where we considered that an infant who has tonic seizures and is also having focal seizures to have EIEE-plus even though focal seizure are accepted as part of the commonly-used EIEE definition. In practice, the opposite sometimes occurs; that is, the syndrome definitions are often applied slightly more broadly than defined, and presence of additional features can be considered acceptable variations. Ultimately, separate groups were used to allow a detailed and nuanced description of the epilepsy and its evolution, but the groups were collapsed for the purposes of calculating incidences and studying the aetiologies associated with each epileptic syndrome. It remains to be seen whether ascribing a syndrome to those infants with previously unclassified epilepsies is useful for predicting aetiology or epilepsy outcome, but they are certainly of use for clinician- to-clinician communication to allow efficient description and understanding of the electroclinical phenotype.

6.2.2 Age of onset Epilepsy onset occurred at 1 day – 17.3 months (median 4.6 months, IQR 5.4 months) (Figure 6.1). The neonatal period (< 1 month old) was the most common age of epilepsy

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onset. Of 17 infants with neonatal onset of epilepsy, seizures began in the first week of life in 15. Just 12 infants had onset between 12-18 months.

Figure 6.1 Age of epilepsy onset in 114 infants with SEI

6.2.3 Time to presentation The duration from seizure onset to presentation was 0 days – 1.1 years (median 13 days, IQR 1.4 months). 83 infants presented within one month of seizure onset, 18 within 1-3 months and 13 after a delay of more than three months. 30 infants presented on the day of the first seizure. Five infants had a delay to presentation of more than six months, all of whom had spasms (4) and/or myoclonic seizures (2) at epilepsy onset.

6.2.4 Seizure types All seizures: 47 (41%) infants had one seizure type in the first two years of life, 28 had two and 33 had three or more. The number of seizure types was unclear in six infants. Of these, five had at least one type, having multiple semiologies reported without definite distinct sites of onset either clinically or electrically. The sixth had at least two types, being spasms and at least one type of focal seizures. More than one seizure type was present at epilepsy onset in 35 infants. 44 infants had an evolution of seizure types.

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Table 6.1 lists the seizure types present at onset and evolution of the epilepsy using the 2016 ILAE seizure classification (with modifications as above).Appendix J lists the seizure types in each infant according to the 1981, 2010 and 2016 ILAE seizure type classifications.

Table 6.1 Seizure types present at onset and evolution of the epilepsy in 114 infants with SEI

Number of infants

Onset Evolution Total

Epileptic spasms 53 35 74

Focal – motor 48 18 49

Focal – non-motor 20 8 24

Generalised – tonic 6 9 15

Generalised – atonic 0 1 1

Generalised – clonic 0 0 0

Generalised – tonic-clonic 0 1 1

Generalised – clonic-tonic-clonic 0 0 0

Generalised – myoclonic 7 12 16

Generalised – myoclonic-atonic 0 1 1

Generalised – absence (typical) 1 2 3

Generalised – absence (atypical) 0 1 1

Generalised – absence (myoclonic) 0 1 1

Generalised – absence (eyelid myoclonia) 0 0 0 Seizure types classified using the modified 2016 classification

Spasms: Epileptic spasms were the most common seizure type. Spasms occurred in 74 (65%) infants, at onset of the epilepsy in 53 and beginning at evolution of the epilepsy in 21. Forty (54%) infants with spasms had other seizure types, most commonly focal seizures.

Focal seizures: Focal seizures occurred in 61 (54%) infants, with more than one focal seizure type in 31 (with a further six infants possibly having more than one seizure type). 49 infants had focal seizures with predominantly motor features and 24 with predominantly non-motor features. Most infants with focal seizures had impaired

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awareness; eight infants had ‘simple partial’ seizures, 49 ‘complex partial’, one ‘partial  generalised tonic-clonic’ and one ‘partial – unclear’. 56 infants had their first focal seizures at epilepsy onset, and five after an evolution of the epilepsy. 41 infants with focal seizures had other seizure types, most commonly epileptic spasms.

Generalised seizures: Generalised seizure types (non-spasms) occurred in 31 (27%), with eight infants having more than one type. Generalised tonic seizures occurred in 15, tonic-clonic in one, clonic in none, myoclonic seizures in 17 (myoclonic in 16, myoclonic-atonic in one), absence seizures in five (typical absence in three, atypical absence in one, myoclonic absence in one) and atonic in one. 14 infants had generalized seizure types at epilepsy onset, and 17 following an evolution of the epilepsy. 22 infants also had focal seizures and/or spasms.

6.2.5 EEG Interictal EEG: 107 (94%) infants had an EEG at presentation (defined as EEG at within two weeks after initial presentation with epilepsy, while seizures were ongoing, and before any evolution of the epilepsy), six had their first EEG after their epilepsy had evolved and one after a spontaneous resolution of epileptic spasms. At epilepsy onset, 102 infants had an abnormal interictal EEG, with IEDs present in 101. Burst- suppression was seen in nine, hypsarrhythmia or modified hypsarrhythmia in 39, generalized spike-wave in four, unifocal IEDs in 15, multifocal IEDs in 33 and both generalized and focal epileptiform activity in one. One infant had a generalized background slowing without IEDs. The interictal EEG evolved in 41 infants with ongoing seizures, with hypsarrrhythmia or modified hypsarrhythmia in 15, generalized spike-wave in nine, generalized electrodecrements in one, unifocal IEDs in four, multifocal IEDs in six and a generalized background slowing without IEDs in four. Four infants with an evolution of seizure types had no change in their interictal EEG (multifocal IEDs in two, unifocal IEDs in one, generalized spike-wave in one). The EEG findings following seizure evolution were unknown in three infants (one had no further EEGs, two had EEG studies outside of Victoria that were not available for review). 112 infants had IEDs recorded on EEG before 18 months old; the remaining two had seizures recorded but no IEDs.

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Ictal EEG: 82 (72%) infants had seizures recorded on EEG, 49 at epilepsy onset, 17 at epilepsy evolution and 16 at both onset and evolution. 44 infants had more than one type of seizure recorded. Epileptic spasms were recorded in 44, focal motor seizures in 30, focal non-motor seizures in 13, generalized tonic seizures in 13, generalised atonic seizures in one, generalized myoclonic seizures in 10, generalised myoclonic-atonic seizures in one, generalised typical absence seizures in three and generalised atypical absence seizures in one. 41 infants had spasm complexes, 40 had one or more types of focal ictal rhythm, 14 had generalised spike-wave and 14 generalised electrodecrements. One infant had generalised tonic seizures without an obvious ictal correlate. One infant had spasms recorded on an EEG performed outside of Victoria that was not available for review.

6.2.6 Seizure frequency 109 infants had multiple daily seizures for at least a week, many continuing at this frequency for considerably longer. Seizure frequency in the four infants with the least frequent seizures was multiple weekly, these infants having days with multiple seizures interspersed with seizure free days.

6.2.7 Epileptic syndromes Epilepsy onset: The epileptic syndromes at onset are presented in Table 6.2. Using the definitions of prototypic and variant syndromes noted in Table 3.4, an epileptic syndrome was allocated to all infants. Details of how these syndrome definitions were applied in each infant are provided in Appendix K, and specific examples with interictal and ictal EEGs are provided for some infants in Appendix L. West syndrome and its variants, namely infantile spasms without hypsarrhythmia and/or with additional seizure types, were the most common syndromes, seen in 52 (46%). Fourteen (27%) of these infants had a variant syndrome, rather than prototypic West syndrome, having an EEG that was not classically hypsarrhythmic and/or additional seizure types. All but one infant with epileptic spasms was considered to have West syndrome or a variant; the remaining infant having ‘EIMFS-plus’. The majority of the remaining infants had focal epilepsies, including well-described syndromes such as EIMFS, and other uni- and multifocal epilepsies. No infant had the syndrome MENPD; all the other ILAE ‘severe’ epileptic syndromes of infancy were seen in at least one infant.

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Table 6.2 Epileptic syndromes at epilepsy onset

Syndrome at onset Number Percent Variant features

EIEE 2 N/A EIEE-like 5 Focal tonic seizures in four, no tonic seizures in one EIEE-plus 1 Additional seizure type – focal seizures in one All EIEE variants 8 7

EME 1 N/A EME-like 1 EEG not burst suppression (discontinuous only) in one EME-plus 0 N/A All EME variants 2 2

EIMFS 8 N/A EIMFS-like 1 Independent left and right seizure onsets not confirmed (but intra-ictal activation of an independent contralateral rhythm recorded, and multiple seizure semiologies described) in one EIMFS-plus 1 Additional seizure type – spasms in one All EIMFS variants 10 9

West syndrome 38 N/A West syndrome-like 8 EEG not hypsarrhythmic in eight West syndrome plus 3 Additional seizure types – myoclonic seizures in two, focal seizures in one West syndrome-like-plus 3 EEG not hypsarrhythmic and additional seizure type – focal seizures in three All West syndrome variants 52 46

Dravet syndrome 0 N/A Dravet syndrome-like 7 Later seizure types not present in five, later seizure types not present and early onset in one, later seizure types not present, early onset and development abnormal prior to seizure onset in one All Dravet syndrome variants 7 6

MENPD 0 N/A All MENPD variants 0 0

LGS 0 N/A LGS-like 3 No slow spike-wave and onset < 1 year old in two, no slow spike-wave in one All LGS variants 3 3

MAE 0 N/A MAE-like 2 Abnormal development before seizure onset in two

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Syndrome at onset Number Percent Variant features

BMEI-like 1 Pharmacoresistant in one CAE-like 1 Early onset in one GGE-other 0 N/A All (non-Dravet syndrome) 4 3 GGE variants

Focal-uni 12 N/A Focal-other 11 N/A Focal-multi 5 N/A All (non-named syndrome) 28 25 focal epilepsies Total 114 100

BMEI = benign myoclonic epilepsy of infancy, CAE = childhood absence epilepsy, EIEE = early infantile epileptic encephalopathy, EIMFS = epilepsy of infancy with migrating focal seizures, EME = early myoclonic encephalopathy, focal-uni = unifocal epilepsy, focal-multi = multifocal epilepsy, focal- other = other focal epilepsies, GGE = genetic generalised epilepsy, LGS = Lennox Gastaut syndrome, MAE = epilepsy with myoclonic-atonic seizures, MENPD = myoclonic encephalopathy in a non- progressive disorder, N/A = not applicable

Epilepsy evolution: An evolution of the epilepsy, defined as evolution to another epileptic syndrome or emergence of new seizure types without syndrome evolution, occurred before two years old in 48 (42%) infants. Of these, 44 had at least one new seizure type, and four had only a change in the interictal EEG. The epileptic syndrome evolved in 45 of these 48 infants. The three infants with new seizure types but no change to the epileptic syndrome were two infants with Dravet syndrome-like epilepsy and one with MAE-like epilepsy. In the remaining 66 infants, seizures had ceased in 39 and were persistent in 27 at two years old or death. Evolution occurred in more than half of infants without an archetypal syndrome with focal epilepsy at onset and in approximately 1/3 of those with West syndrome or a West-variant at onset. Evolution was less commonly seen in the neonatal and early infant-onset epileptic syndromes, perhaps due to high rates of mortality in infancy (discussed later in chapter), prior to any evolution that may have otherwise occurred. The most common epileptic syndromes at evolution were West syndrome and its variants, occurring in 26, and LGS and variants in 12. In contrast, only one infant evolved to a focal epilepsy. All of the infants with a Dravet syndrome -like phenotype at onset had an evolution of their epilepsy, four to Dravet syndrome, two whose epilepsy remained Dravet syndrome -like

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but who had new seizure types develop, and one to West syndrome (left and right hemiclonic seizures evolving to epileptic spasms). The evolution and persistence of seizures by epileptic syndrome is further described in the ‘Seizure treatment and outcome’ section below.

Incidence of epileptic syndromes at presentation and evolution: The incidence of each epileptic syndrome in Victoria is noted in Table 6.3. Here, the incidence of infantile spasms (seizure type) is 33.2/100,000 live births/year (74 infants), and of West syndrome and its variants (epileptic syndrome) is 32.8/100,000 live births/year (73 infants), or approximately 1:3000. The incidence of infantile spasms with hypsarrhythmia (West syndrome and West syndrome +) is 26/100,000 live births/year (58 infants – West syndrome / West syndrome + beginning in 43 infants at epilepsy onset and in 15 at epilepsy evolution).

The estimated incidence of Dravet syndrome and Dravet syndrome -like epilepsies in Victoria is 5.8/100,000 live births/year, this figure including infants who did not meet the study definition of SEI (see Chapter 5). Of the infants who fell within the inclusion criteria for SEI, six ultimately had a syndrome diagnosis of Dravet syndrome or Dravet syndrome -like. One infant’s epilepsy was Dravet syndrome-like at onset but evolved to West syndrome. Given this evolution was not typical of either Dravet syndrome or similar variants, this infant was not included in the Dravet syndrome incidence figures. The incidence of Dravet syndrome and Dravet syndrome-like epilepsies meeting SEI inclusion criteria is 2.7/100,000 live births/year, making up approximately half of the estimated Dravet syndrome / Dravet syndrome -like epilepsies in Victoria. The incidence of infantile spasms and the estimated incidence of Dravet syndrome in Victoria are similar to previous reports, although may be underestimated here due to the study methodology (Bayat et al., 2015; Cowan & Hudson, 1991; Wu et al., 2015).

In this study, the incidence of EIMFS and EIEE were 4.5/100,000 live births/year and 3.6/100,000 live births/year respectively, these being of a similar order of magnitude to the incidence of Dravet syndrome.

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Table 6.3 Incidence of ILAE neonatal and infantile severe epileptic syndromes (and their variants)

95% Incidence confidence (per 100,000 Incidence Previous Epileptic syndrome N interval (per live (1:X000) studies 100,000 live births/year) births/year)

Early infantile epileptic 8 3.6 1.6-5.6 1:28,000 - encephalopathy

Early myoclonic 2 0.9 0-4.9 1:111,000 - encephalopathy

0.55- Epilepsy of infancy with 1.6/100,000 10 4.5 8.1-11.9 1:22,500 migrating focal seizures live births/year-

West syndrome/ infantile 25-42/100,000 74 33.2 31.9-34.5 1:3000 spasms live births/year

1:15,700- Dravet syndrome** 13** 5.8 4.1-7.5 1:17,500 1:22,000

**Infants with Dravet syndrome and Dravet syndrome -like phenotypes identified through the screening sources and determined to not meet SEI criteria are included in this estimate of Dravet syndrome incidence, given that the study inclusion criteria clearly meant that not all infants with Dravet syndrome would meet our definition of SEI. See chapter X (Ascertainment and epidemiology) for further detail.

Aetiology by epileptic syndrome: The aetiology was known in over half of infants with each epileptic syndrome, with the exception of those with ‘focal – other’ and ‘focal – multi’ epilepsies. Almost all infants with unifocal epilepsy at onset had a structural (malformative or acquired) basis, as did approximately 50% of infants with West syndrome and variants. 36/74 (49%) infants who had West syndrome or a variant at onset or at evolution had a structural aetiology (12 acquired (16%) and 24 (33%) malformative). Structural aetiologies were uncommon in other epileptic syndromes, and no infant with EIEE or variants had a structural cause. Infants with known non- structural aetiologies had predominantly single gene disorders for most syndromes, with a smaller number having metabolic or chromosomal causes. West syndrome was an exception, with chromosomal causes (predominantly Trisomy 21) more commonly identified than single gene aetiologies.

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Table 6.4 Aetiologies by epileptic syndrome at epilepsy onset

Aetiology Epileptic Aetiology syndrome at N Acquired Brain Metabolic Chromo- Single List known? onset malform- somal gene ation disorders

Molybdenum cofactor deficiency in 1, 8 6 (75%) 0 0 2 0 4 EIEE mitochondrial in 1, KCNQ2 in 2, SCN2A in 2

PNPO deficiency in 1, Wolf-Hirschhorn syndrome 2 2 (100%) 0 0 1 1 0 EME in 1

PMG in 1, mitochondrial in 1, KCNT1 in 1, 10 6 (60%) 0 1 1 0 4 EIMFS SCN8A in 1, TBC1D24 in 1, KCNQ2 in 1

HIE in 4, stroke in 2, PVL in 2, complicated meningitis in 1, ischaemic injury (mechanism

181 unknown) in 1, FCD in 8, TS in 2, PMG in 2,

West syndrome 52 34 (65%) 11 15* 0 6 2* malformation of cortical development (other) in 2, lissencephaly in 1, Aicardi syndrome in 1, T21 in 5, Chr15q21.3q22.2 del in 1, NSD1 in 1, SMC1A in 1

Dravet HIE and hypoglycaemia in 1, SCN1A in 3, Chr 7 5 (71%) 1 0 0 1 3** syndrome 2q24.3 del (incl. SCN1A and SCN2A) in 1

Prematurity and PVL in 1, Chr 15q21.3q22.2 4 3 (75%) 1 0 0 1 1 Other GGE deletion in 1, SYNGAP1 in 1

LGS 3 2 (67%) 1 0 1 0 0 HIE in 1, Tay-Sachs disease in 1

Complicated meningitis in 1, FCD in 6, TS in 2, Focal (uni) 12 11 (92%) 1 9*** 1 0 0 Sturge Weber in 1, mitochondrial in 1

Aetiology Epileptic Aetiology syndrome at N Acquired Brain Metabolic Chromo- Single List known? onset malform- somal gene ation disorders

Lissencephaly in 1, complex malformation of cortical development in 1, pontocerebellar 11 5 (45%) 0 3**** 0 0 2 Focal (other) hypoplasia in 1, Aicardi-Goutières syndrome in 1, SCN8A in 1

Focal (multi) 5 2 (40%) 0 2 0 0 0 TS in 1, achondroplasia in 1

Total 114 76 (67%) 14 31 6 9 16 In addition to the above listed infants, infants with suspected aetiologies were:

182 *4 infants with suspected FCD and 1 with suspected AARS

** 1 infant with suspected SCN1A ***1 infant with suspected FCD

****1 infant with suspected FCD

6.2.8 Seizure treatment and outcome All infants: All but one infant was treated with antiepileptic drugs (AEDs). The median number of AEDs used up to two years old was four (range 0-13, IQR 4). Seven infants had tried a ketogenic diet and two the Modified Atkins diet. Five infants had had six epilepsy surgeries (tuberectomies (2 in 1 infant), sublobar resection in 2, multilobar resection in 2). No infant had had a vagus nerve stimulator inserted.

Nineteen (17%) infants were seizure free within a month of initial presentation and treatment. Twelve infants had ongoing seizures for 1-3 months and seven for 3-6 months before becoming seizure free. At two years old, 46 (40%) infants had ongoing seizures. In surviving infants, spasms were ongoing in 19/71 (27%), focal seizures in 25/47 (55%) and generalized seizures other than spasms in 17/26 (65%) at two years old. 30 (41%) infants with a history of spasms had ongoing seizures of any type at 2 years old. Of the 50 (44%) infants who were seizure free, 25 remained on AEDs. 17/18 deceased infants had ongoing seizures at the time of their death.

No difference was seen in the number of infants with ongoing seizures until two years old or death with respect to their age of seizure onset (<6m 41/71 (58%), 6-12m 15/31 (48%) and >12m 7/12 (58%)) (χ2 = 0.816, df = 2, p = 0.665) or time from seizure onset to presentation (<1m 49/83 (59%), 1-3m 9/18 (50%), >3m 6/13 (46%)) (χ2 = 1.08, df = 2, p = 0.581).

Infants with spasms: The first AED used for spasms was prednisolone in 29 (39%), vigabatrin in 15 (20%), prednisolone and vigabatrin in three (4%) (participants in the International Collaborative Infantile Spasms Study (O'Callaghan et al., 2017), levetiracetam in nine, valproate in seven, clonazepam in two, clobazam in one, phenobarbitone in one, phenytoin in one, topiramate in one, pyridoxine in one, and epilepsy surgery in one (who had spasm onset shortly before planned surgery for refractory focal seizures). The first AED was not known in two. One infant had no treatment of spasms, as there had been spontaneous resolution prior to presentation.

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Spasms ceased in 36/73 (49%) treated infants after the first AED used to treat the spasms, later recurring in 10 of these infants. Table 6.5 lists the treatment response to the first AED used to treat spasms in each infant.

Five patients had an indication for use of vigabatrin as first line treatment for spasms (all had TS); the first agent used in these infants was vigabatrin in three, prednisolone in one (NB diagnosis of TS was delayed in this infant) and unknown in one. One had a relative contraindication to prednisolone (immunodeficiency); thus, vigabatrin was given first, with prednisolone used later because of ongoing spasms. No other infants had a contraindication to use of steroids, or an indication for another agent as first-line therapy. Overall, only 37 of 73 infants (51%) treated for infantile spasms received what could be considered evidence-based first-line treatment, these being 29 infants treated with prednisolone, three infants treated with prednisolone and vigabatrin, four infants treated with vigabatrin (three TS, one with a relative contraindication to prednisolone) and one with epilepsy surgery. .Resolution of spasms was seen in 32/37 infants (86%) who had evidence-based first-line treatment, with later recurrence in nine. Thus, 23/37 (62%) who had evidence-based first-line treatment had persistent freedom from spasms. 4/36 infants (11%) who received other first-line treatments had spasm resolution, with later recurrence in one. Therefore, 3/36 (8%) with non-evidence-based first-line treatment had persistent freedom from spasms.

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Table 6.5 Effect of the first antiepileptic agent used to treat spasms

First therapy for epileptic spasms Spasms resolved with PNL+VGB first agent? Epilepsy PNL (ICISS VGB LEV VPA Other Unknown Total surgery trial)

Yes 18 2 4 1 0 0 1 0 27

Yes (but 6 1 3 0 0 0 0 0 10 recurred)

No 5 0 8 0 9 7 6 2 35

Total 29 3 15 1 9 7 7# 2 73^

^ One infant with spontaneous resolution of spasms before presentation did not receive treatment and is not included in this table # clonazepam in 2, clobazam in 1, phenobarbitone in 1, phenytoin in 1, pyridoxine in 1, topiramate in 1 PNL = prednisolone, VGB = vigabatrin, ICISS = International Collaborative Infantile Spasms Study, LEV – levetiracetam, VPA = valproate

25/47 (53%) infants receiving ongoing treatment for failure of the initial therapy (lack of resolution (37) or resolution followed by relapse (10)) had spasm resolution with a second or subsequent antiepileptic drug, being prednisolone in 10, ACTH in one, vigabatrin in eight, topiramate in three, clonazepam in one, levetiracetam in one and lamotrigine + valproate in one. 6/25 had a later spasm recurrence (two who were treated with prednisolone, three with vigabatrin and one with levetiracetam).

Before two years old, 51 (70%) infants were given corticosteroids (prednisolone in 50, ACTH in one, used as 1st-4th agent) and 39 (53%) vigabatrin (1st-7th agent) for treatment of spasms. Nine received neither prednisolone nor vigabatrin. Steroids resolved spasms in 38/51 (75%), with persistent freedom from spasms in 29 (57%) and recurrence in nine. Vigabatrin resolved spasms in 17/39 (44%), with persistent freedom from spasms in 11 (28%) and later recurrence in six.

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Three infants had epilepsy surgery for refractory spasms (+/- other seizure types). Following surgery, one was seizure free, a second had ongoing focal seizures and a later spasm recurrence and the third had a 9-month seizure-free period before spasm recurrence. Three infants were given a ketogenic diet for refractory spasms. One had an initial benefit that was not sustained, one had no benefit and the effect was unknown in the third.

Infants with focal seizures: The AEDs used to treat focal seizures were highly variable, as was the order in which they were given. Persistent freedom from focal seizures was reported with an antiepileptic drug in 12, being levetiracetam in four, vigabatrin in three, phenobarbitone in two, valproate in one and lamotrigine + valproate in one.

6/13 had later development of spasms (5) or myoclonic-atonic seizures (1), and one had concurrent spasms that were ongoing following resolution of focal seizures. These infants had received levetiracetam (4), valproate (1), carbamazepine (1) and phenobarbitone (1) for their focal seizures. Two infants had cessation of focal seizures, which later recurred (vigabatrin and oxcarbazepine in one, levetiracetam in one). Three infants had a dose-related response to an AED (phenytoin in two, oxcarbazepine in one, these infants having SCN8A (2) and SCN2A mutations (1)), having periods of seizure freedom interspersed with periods of seizure recurrence when drug levels were low (phenytoin) or the infant ‘grew out of’ the dose (oxcarbazepine), with consistent seizure resolution upon dose increase. One infant had an apparently effective drug ceased due to side effects (valproate, first drug used, seizure free for two weeks, developed hyperammonaemia). Four infants became seizure free following epilepsy surgery, but focal seizures later recurred in one. No infant was seizure free on a ketogenic or modified Atkins diet.

Infants with generalized seizures other than spasms: AED treatment of generalized seizures was also highly variable. Persistent seizure freedom (from myoclonic seizures in one and tonic seizures in three) was reported with an antiepileptic drug in four, being prednisolone in one, ACTH in one, vigabatrin in one and phenobarbitone + clonazepam in one. Three infants also had spasms, and had developed tonic or myoclonic seizures

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prior to successful treatment of spasms. Both the generalized seizure type and spasms ceased with the same drug. Two infants had a dose-related response to an AED (pyridoxal-5-phosphate in one with PNPO deficiency, phenytoin in one with an SCN2A mutation). One infant had resolution of myoclonic-atonic seizures following epilepsy surgery. No infant was seizure free on a ketogenic or modified Atkins diet.

By epileptic syndrome: Variability was seen in the seizure outcomes at two years old between the different epileptic syndromes at seizure onset (Table 6.6, Figure 6.2). Seizure cessation, whether on or off AEDs was seen in almost 33/74 (45%) of infants with West syndrome and variants and 11/28 (39%) of infants with focal epilepsies, but not seen in any infant with a Dravet syndrome -like presentation. Few infants with neonatal and early infantile-onset epileptic syndromes were seizure free at two years old, but it is notable that a significant proportion of these infants were deceased by this age. All three seizure-free infants with EIEE or EIMFS (and variants) at onset had KCNQ2 mutations.

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Figure 6.2 Seizure outcome at two years old by epileptic syndrome at onset

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*1 infant, off all standard antiepileptic drugs but on pyridoxal-5-phosphate

Evolution of the epilepsy before two years old was seen in 36/63 (57%) of infants with ongoing seizures at two years old or death, and 12/51 (24%) of those whose seizures had ceased by two years old or death.

Table 6.6 Epilepsy evolution and seizure outcome at two years old or death by epileptic syndrome at onset

Epilepsy evolution No epilepsy evolution Epilepsy syndrome at onset Seizures Seizures Deceased Deceased ceased ongoing ceased ongoing

EIEE 1 1

EIEE-like WS-like 1 LGS-like 1 1 2

EIEE-plus 1

EME LGS-like 1

EME-like 1

EME-plus WS+ 1, WS- WS+ 1 1 1 3 EIMFS like + 1

EIMFS-like 1

EIMFS-plus 1 LGS-like 5, WS-like 2, WS LGS-like 2 WS-like+ 2, 23 2 LGS 1, MAE-like 1 Focal (uni)1, WS-like+ 1 4 1 WS-like LGS-like 1

WS-plus WS-like+1 1 1

WS-like- 2 1 plus DS DS 4, DS-like DS-like 1 DS-like 1, WS 1

MENPD

LGS

LGS-like 1 2

MAE

MAE-like MAE-like 1 1

BMEI-like 1

CAE-like 1

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Epilepsy evolution No epilepsy evolution Epilepsy syndrome at onset Seizures Seizures Deceased Deceased ceased ongoing ceased ongoing

GGE-other WS-like+ 2, WS+ 4, WS 1, WS 1, WS- 1 1 Focal-uni MAE-like 1 like 1 WS 1, LGS- WS 1 2 3 3 Focal-other like 1

Focal-multi WS+ 3 WS-like+ 1 1 Epilepsy evolution is defined as evolution of the epileptic syndrome or emergence of new seizure types without syndrome evolution before two years old. DS = Dravet syndrome, WS = West syndrome.

By aetiology: There was variation in the proportion of surviving infants with ongoing seizures at two years old according to aetiology. 4/5 (80%) of infants with metabolic aetiologies had ongoing seizures, with just 1/8 (12.5%) with chromosomal aetiologies having ongoing attacks. 47-52% of infants in the other aetiologic categories had ongoing seizures.

Seizures were ongoing at two years old or death in 20/38 (53%) of infants with unknown aetiology and 42/76 (55%) whose aetiology was known.

Evolution of the epileptic syndrome was seen in 17/31 (55%) infants with malformative aetiologies (predominantly to West syndrome and LGS or variants of), but just 2/9 (22%) and 4/14 (29%) of infants with chromosomal and acquired aetiologies showed evolution respectively (Table 6.7).

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Table 6.7 Epilepsy evolution and seizure outcome at two years old by aetiology

Epilepsy evolution No epilepsy evolution

Aetiology Seizures Seizures Deceased Deceased ceased ongoing ceased ongoing

WS 3, 6 4 Acquired LGS 1

WS 7, LGS 3, WS 5 8 2 4 Brain malformations MAE 1, focal (uni) 1

Metabolic LGS 1 WS 2 2 1

Chromosomal LGS 1 DS 1 6 1

DS 3, Single gene LGS 1 6 2 3 disorders MAE 1 WS 4, WS 4, LGS 3, WS 1 12 3 6 Unknown LGS 2 DS 2, MAE 1

Total 12 33 3 38 13 15

Epilepsy evolution is defined as evolution of the epileptic syndrome or emergence of new seizure types without syndrome evolution before two years old. DS = Dravet syndrome, WS = West syndrome.

Non-pharmacologic treatments before and after two years old: Nine infants had epilepsy surgery after two years old, bringing the total number of infants who had surgery to 14. Of these 14, seven were seizure free at last review, two with TS had resolution of the operated seizure type but ongoing (infrequent) focal seizures of another type, two were seizure free for nine months then had seizure recurrence, two had ongoing seizures and one had unknown outcome (surgery one week before the time of writing).

A further six infants tried the ketogenic diet by two years old, and 13 by the time of writing. Of these 13, one was seizure free, three had a clinically significant seizure reduction (defined as >30% reduction in seizures, or note of clear improvement in

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seizure frequency where percentage reduction was not documented) and continued on the diet, and three had an initial benefit that was not sustained, so the diet was ceased.

6.2.9 Commentary The distribution of ages of seizure onset in this group with SEI mirrors that of all epilepsies in the first two years of life , in that the largest proportion of the group has seizure onset in the first ~ six months of life, this proportion declining with increasing age (Chevrie & Aicardi, 1977; E. Gaily et al., 2016). In our study, when considered month-by-month, the first month of life is the most common onset age. The infants with onset at this age are predominantly those epilepsies with EEG features of burst- suppression (EIEE and EME) and multifocal discharges (EIMFS and ‘focal – multi’). This has perhaps been underappreciated previously as the incidence of these epilepsies has not been previously reported and, in the past neonatal seizures were not considered epilepsy meaning that many epidemiologic studies of infantile-onset epilepsy excluded neonates. Over a third of infants had onset between 3-7 months old; this being expected as it is the peak age of onset for the most common seizure type, epileptic spasms (Hrachovy & Frost, 2013).

As expected, the time between first seizure and presentation to medical attention was brief in most infants, probably because of high seizure frequency and the large number of infants with focal seizures. Urgent medical attention (e.g. ambulance and emergency department) is commonly sought for focal seizures, particularly those with motor features. The time to presentation was more varied for infants with epileptic spasms as these are sometimes subtle or mistaken for non-epileptic events. Nevertheless, the almost inevitable associated change in developmental trajectory prompts medical presentation even if the seizures themselves do not, inevitably leading to diagnosis, albeit sometimes delayed. A number of studies have shown poorer developmental outcomes in infants with delayed presentation and/or treatment (Berg et al., 2014; Cormack et al., 2007; D'Argenzio et al., 2011; Eisermann et al., 2003; Jonas et al., 2004; Kivity et al., 2004; Loddenkemper et al., 2007; Matsumoto et al., 1981; O'Callaghan et al., 2011). The number of such infants in our group is probably too small, and the developmental levels and long term outcome not measured precisely enough, to determine if that is the case here.

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The seizure burden in infants with SEI is extremely high, with almost all infants having multiple daily seizures at some point in their epilepsy, some continuing at this frequency for months or years. Over half had more than one seizure type, these infants being predominantly those with both spasms and focal seizures, and those with multiple types of focal seizures.

As expected and consistent with previous reports, epileptic spasms were the most common seizure type. It has been previously reported in infants with refractory seizures that four seizure types, spasms, and seizures with tonic, clonic or hypomotor semiology, accounted for over 80% of all seizures (Hamer et al., 1999). It is not clear from that study whether the ‘tonic’ and ‘clonic’ seizures reported were generalized or focal. In this study, seizures with these semiologies were common, the clonic seizures being almost universally focal, whereas both focal and generalized tonic seizures were seen. As in that study, other (mainly generalised) seizure types, such as myoclonic, atonic and absence seizures were uncommon here. The low rate of generalised seizure types such as generalized tonic-clonic, myoclonic and absence seizures is in contrast to older age groups where these are relatively common seizure types (rev in (Bureau et al.)), this presumably reflecting differences in the brain networks engaged by and during seizures at different ages.

The interictal EEG was abnormal with IEDs in most infants at onset. In this study, the five infants with normal EEGs at onset had GGE phenotypes – four Dravet syndrome - like (three of whom evolved to Dravet syndrome proper) and one with a BMEI-like phenotype. The most common EEG patterns across the group were those that typically accompany spasms and focal seizures, namely hypsarrhythmia/modified hypsarrhythmia, focal and multifocal IEDs.

The interictal EEG findings at evolution were more commonly generalised (hypsarrhythmia, generalised spike wave etc.) or multifocal than unifocal. 58/74 (78%) infants with epileptic spasms had a hypsarrhythmic EEG at some point, not dissimilar to a previous reported figure of 60% (Lacy & Penry, 1976). Ictal EEG recordings were obtained in most patients, often in routine EEGs, reflecting the high seizure frequency seen in most infants.

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West syndrome and variants were the most common epileptic syndromes at onset, and West syndrome and variants and LGS and variants the most common at evolution. This study looked only at evolution up to two years old, by which point the epilepsy had evolved in approximately 40% of infants. The number of infants with an evolution will likely increase with a longer duration of follow-up, and some infants may have a second evolution of their epilepsy. Of particular note, LGS is reported to occur in 15-50% of infants with West syndrome (Dulac et al., 2007; Hrachovy & Frost, 2013; Trevathan et al., 1999; Yamatogi & Ohtahara, 2002). Here, prototypic LGS (rather than LGS-like epilepsy) was seen in only one infant by two years old, likely because the follow-up period did not extend to the age of peak onset of LGS (3-5 years) (rev in (Stephani, 2006)). Certainly, a number of other infants had an LGS-like evolution, which may in time meet the definition of prototypic LGS.

While unifocal epilepsies commonly evolved to other syndromes (mainly West syndrome and variants), evolution from any other syndrome to a unifocal epilepsy was not common. In this study, almost all infants with unifocal epilepsy had a structural aetiology. It may be that, where seizures are ongoing and refractory, the epilepsy is more likely to ‘step up a gear’ to a generalised epileptic syndrome rather than ‘step down’ to a focal epilepsy. An alternative explanation may be that evolutions from West syndrome and its variants to a unifocal epilepsy do occur, but not until an older age.

It has been previously reported that 75% of infants with EIEE evolve to West syndrome (Yamatogi & Ohtahara, 1981, 2002). In this study, this evolution was seen in only 1/8 infants. The reasons for the differences are not known, but may be due to expected variation given our small group size, and heterogeneity of underlying aetiologies with corresponding differences in evolution and outcome.

This study confirms prior estimates of the incidence of infantile spasms and Dravet syndrome, reports an incidence for EIMFS that is considerably higher than previous reports, and reports for the first time the incidences of EIEE and EME (Bayat et al., 2015; Cowan & Hudson, 1991; E. Gaily et al., 2016; McTague et al., 2013; Wu et al.,

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2015). The incidences of EIEE and EIMFS in our study are not much lower than that of Dravet syndrome, perhaps surprisingly as these syndromes are considered to be very rare. The methods used in this study mean that the figures derived for EIEE and EIMFS are not overestimated. The estimated incidence of Dravet syndrome in this study is less robust as not all patients were ascertained within the inclusion criteria, but when put together with all that were identified in the state of Victoria, the incidence is comparable to previous reports. Thus, either EIEE, EIMFS and Dravet syndromes are similarly common, or Dravet syndrome is under-recognised. EME has been observed to be an extremely rare epilepsy, this being confirmed in this study, where the incidence is 1:111,000.

Variability was noted in the aetiologies associated with each epileptic syndrome, in many cases mirroring that in previous reports, but with some notable differences. In this study, no infant with EIEE or variants had a structural aetiology, in contrast with previous reports of malformations in as many as 50% (Yamatogi & Ohtahara, 1981, 2002). Like the earlier discussion of differences in evolution of the epilepsy in EIEE to that previously reported, this may reflect that a large number of aetiologies, many individually uncommon, can cause EIEE, resulting in significant variability in the spread of aetiologies seen in different small cohorts. In infants with spasms, like in the UKISS study, we found that approximately half had a structural basis for their epilepsy. However, the current study differed from UKISS in the proportion of infants who had an acquired structural aetiology versus a brain malformation. Here, approximately 2/3 had a malformation and 1/3 an acquired brain injury; these proportions were reversed in the UKISS study (Osborne et al., 2010). The reasons for this are not clear. It is not likely that there are significantly more perinatal/early postnatal acquired brain injuries in the countries that participated in the UKISS study, as the quality of antenatal, obstetric and paediatric care is similar to that in Victoria. Differences may arise from the fact that one study is population-based and the other not, or that the quality of brain imaging has improved since the UKISS study was published, thereby allowing improved diagnosis of malformations that would have been previously unrecognized. It is likely in the current study that the proportion of infants with spasms who have a malformation is even higher than reported here given a number of infants with unknown aetiology are suspected to harbour an occult FCD; such malformations may be more

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readily detected once myelination is complete, or through use of more sophisticated imaging tools. Given the high proportion of malformations in West syndrome and unifocal epilepsies, infants presenting with these epileptic syndromes whose aetiology is unknown at presentation should be strongly suspected to have a brain malformation, whether obvious or occult. Recognising a malformation in such infants can have major treatment implications, as epilepsy surgery may cure their epilepsy and improve developmental outcomes. Single gene disorders dominated the identified aetiologies for most other syndromes. Interestingly, among infants with West syndrome and variants who had a non-structural aetiology, single gene causes were identified in few infants, with chromosomal causes found more commonly. This finding has implications for the currently used techniques for multigene testing in that the diagnostic yield is considerably lower in West syndrome than in other epilepsies.

A significant proportion of infants in this study had seizures ongoing for many months despite prompt presentation and treatment in most. Just one third of infants had seizure resolution by six months after initial presentation. Over half had ongoing seizures at two years old or until death. Evolution of the epileptic syndrome was common in those with ongoing seizures.

Variability was seen across different seizure types, epileptic syndromes and aetiologies in the rates of seizure freedom, this information being potentially useful for prognostication in infants with particular clinical characteristics. A previous large study of seizure outcomes in all infant epilepsies reported ongoing seizures in 70% at two years old, with seizure freedom more likely in those with normal intellect, those with unknown cause and males (Chevrie & Aicardi, 1978).

In our study, infants with normal intellect were similarly more likely to be seizure free at two years old. However, contrasting from the earlier study, in our study, rates of seizure freedom were similar in infants with known (55%) and unknown (53%) aetiologies. This discrepancy may reflect a different composition of the unknown group across the two studies given some infants with single gene disorders and occult malformative aetiologies may not have received a diagnosis in the earlier study. It may also be due to the fact that the other study included all infants with seizures, including

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those with the ‘benign’ infant epilepsies that have normal development, which are now recognized to have a genetic basis but which had unknown cause at the time that study was conducted. There was no difference between genders in the rates of seizure freedom in this study. There was no discernible reason for the gender discrepancy seen in the previous study, thus it seems more likely to represent variation within statistical limits than to have a biologic basis.

Prior studies have reported spasm resolution in 75% of infants by 12-14 months, and ongoing seizures of any type in approximately 50% at four and five years old (Darke et al., 2010; Lux et al., 2005). The figures in this study were comparable (74% spasm free and 60% seizure free at two years old), although reported at different ages. The proportion of infants with focal seizures or generalized seizures (other than spasms) with ongoing seizures at different ages has not been specifically reported previously. It is interesting to note that the prognosis for seizure freedom across the whole group of infants with SEI is better than the prognosis for normal development (discussed below), although both are poor.

Infants in this study had a median of four antiepileptic drugs used by two years old, a median of >2 AEDs being expected given treatment resistance was an inclusion criterion. The exception to this was infants with spasms, one of whom received no antiepileptic treatment and some just one AED, as spasms was an automatic inclusion criteria. Many infants with ongoing seizures at two years old received additional AEDs over the numbers listed here. Thus, the overall exposure to AEDs was high. The broad range of AEDs used, even among infants with the same epileptic syndrome or underlying aetiology, highlights the lack of an evidence-base for treatment of seizures in most infant epilepsies. A potential issue for future treatment trials is the variability in natural history of the different aetiologies that cause particular epileptic syndromes. For example, in EIEE, those with KCNQ2 encephalopathy typically have remission of seizures by early childhood, whereas seizures are ongoing in those with most other aetiologies (Howell et al., 2015; Saitsu et al., 2016; Stamberger et al., 2016; Weckhuysen et al., 2012). Knowing the aetiology will be vital for differentiating treatment effect versus natural history in epileptic syndrome-based trials.

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One of the few seizure types for which there is evidence-based treatment is infantile spasms, where corticosteroids are first-line treatment for most (except for TS, in which vigabatrin is considered first-line), and are associated with improved developmental outcome, at least in infants with unknown cause for spasms (Darke et al., 2010). Despite this evidence, and despite the fact that this study was conducted after that data was published, only half of infants in this study received evidence-based first-line treatment for their spasms. Those who received evidenced-based first-line treatment had substantially higher rates of spasm cessation than those that did not. This is clearly an area in which current practice can be improved at Victorian centres.

For other seizure types, there is not a clear evidence base on which treatments should be first-line (Wilmshurst et al., 2015). A number of AEDs were reported effective in infants with focal or generalized (other than spasms) seizures, although without a clear impression that some drugs were more effective than others at a group level. As previously reported, sodium channel blockers ceased seizures in some infants with SCN2A and SCN8A mutations in this study, with seizure recurrence when drug levels or dose per kilogram reduced (Howell et al., 2015; Larsen et al., 2015). As is well established in Dravet syndrome (Guerrini et al., 1998), some infants with Dravet syndrome in this study had seizure exacerbations with sodium channel blockers, and improved with AEDs such as sodium valproate, although no infant was seizure free.

On the flip side, the study did provide a clearer impression of lack of efficacy of some AEDs in some settings. Levetiractam, one of the most commonly used AEDs in infants in our centres, was not of benefit for epileptic spasms. Spasms did not cease in any of the nine infants given levetiracetam first-line, and persistent spasm cessation was seen in only one infant given levetiracetam as a second or subsequent treatment. Additionally, while focal seizures ceased with levetiracetam in four infants (all with structural aetiologies), concurrent spasms in one infant continued, and the other three subsequently developed spasms while on levetiracetam, suggesting that levetiracetam may not have the ‘preventative’ effect on spasms that can be seen with vigabatrin (Jozwiak et al., 2011). This finding corroborates that of a recent study showing poor efficacy of ‘nonstandard’ AEDs (i.e. other than steroids and vigabatrin) in infantile spasms (Knupp et al., 2016).

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Given the relationship of seizure cessation to developmental outcomes, and the knowledge that AEDs can contribute to developmental impairments and produce other side effects in some infants, treatment trials are needed in order to determine which treatments are more efficacious and which are better tolerated. This needs to be considered both at the larger group level, and within subgroups of infants with particular epileptic syndromes and aetiologies and will inform the order of selection of AEDs.

Non-pharmacologic treatments can have significant benefit in some infants with seizures refractory to AEDs. The RCH has a large epilepsy surgery program with significant experience in young children. Operated infants had refractory seizures, often with spasms, an encephalopathic EEG and developmental arrest/decline. Surgery produced benefit with regards to the epilepsy and/or development in most cases. The literature around the impact of curative epilepsy surgery on developmental outcome shows the most benefit in infants (Loddenkemper et al., 2007), highlighting the need to diagnose brain malformations as early as possible, and consider these infants for an epilepsy surgery work-up in infancy. Ideally, infants with refractory seizures and a deviation in developmental trajectory whose epilepsy is surgically-remediable would have surgery performed in the infant period

The ketogenic diet was of ongoing benefit in approximately one quarter of infant in whom it was trialled, with seizure freedom in only one. Overall, the ketogenic diet was associated with less benefit than surgery, although it provides a potentially efficacious treatment for a different patient group (i.e. those who are not surgical candidates) in whom AEDs have failed. There are some aetiologies for which the ketogenic diet is the treatment of choice (glucose transporter 1 (Glut1) deficiency, pyruvate dehydrogenase deficiency) (Klepper et al., 2005; Wexler et al., 1997). Additionally, the literature would suggest there are subgroups of infants who are more likely to respond to the ketogenic diet, including infants with epileptic spasms and with GGEs such as absence epilepsy (R. H. Caraballo et al., 2005; Kossoff et al., 2002; Thammongkol et al., 2012; van der Louw et al., 2016). The diet should be considered in such infants if the epilepsy is refractory.

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6.3 Development

6.3.1 Pre-seizure onset Prior to seizure onset, development was normal in 44 (39%), abnormal in 53 (46%) and unknown in 17 (15%) who had seizure onset in the first month of life. Assessment of development in the first month of life can be unreliable for a number of reasons. The developmental repertoire of neonates is limited; thus, deviations from normal can be subtle and are not always detected, particularly by first-time parents.

6.3.2 Developmental plateau and regression 96 infants had at least one period of developmental plateau or regression, most commonly coinciding with seizure onset or evolution to epileptic spasms. Plateau or regression was not possible in five infants who died in the neonatal period, and not documented in five. Eight infants had no plateau or regression, as determined from my clinical assessment in one and documented in the medical records in seven. Many infants were reported to have a developmental acceleration after seizure cessation (particularly those with spasms), but this was not documented in all infants (and therefore the proportion of infants in which this phenomenon was present could not be determined) and the magnitude of this change is difficult to quantify.

6.3.3 Developmental outcome All infants: In 96 surviving infants at two years old, development was normal in 10 (10%) and abnormal in 86 (90%), being severe-profound global developmental delay in 41, mild-moderate global developmental delay in in 28, mild-moderate language delay in 16 and mild-moderate motor delay in one.

For 90/96 surviving children, information was available on the specific skills attained by two years old. Of the remaining six, two had limited information on specific skills attained, and four were lost to follow up before age two years. All six had global developmental delay, being mild-moderate in four and severe-profound in two at last review.

For the 90 infants with detailed information on milestones at 2 years, eleven had normal expressive language development, with at least 50 words and two-word phrases. 25

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infants spoke at least five words. 33 infants were not able to babble. 26 infants had normal motor development at two years old, and 41 were independently ambulant. 33 infants were unable to sit. 65 infants demonstrated good visual attention. Just one infant had an autism spectrum disorder diagnosed by two years old.

13/18 infants deceased before two years old had severe-profound global developmental delay. 10/13 were not visually attentive. Eleven had no head control, one had head control and one was able to roll. All but one (who had sensorineural hearing loss) demonstrated some response to voice. Seven infants were able to coo, six had no vocalization. Supplemental feeding was required in all 13. 5/18 died in the neonatal period and thus developmental level was not able to be determined.

Severe-profound developmental delay (or non-assessable development for those with neonatal death) at two years old or at death was highest in those with younger age at onset, being present in 17/17 (100%) of infants with seizure onset under one month old, 12/21 (57%) with onset at 1-3 months, 11/33 (33%) with onset at 3-6 months, 13/31 (42%) with onset at 6-12 months and 6/12 (50%) with onset after 12 months old (χ2 = 21.8, df = 4, p = <0.001). Severe-profound delay at two years old or death was higher in those with prompt presentation, with 48/79 (61%) infants who presented to medical attention within one month of seizure onset having severe-profound delay, compared with 6/22 (27%) who presented after 1-3 months and 5/13 (38%) who presented after more than three months (χ2 = 8.77, df = 2, p = 0.012). 45/63 (71%) infants with ongoing seizures at two years old or death had severe-profound (or non-assessable for neonatal deaths) developmental delay, as did 14/51 (27%) of infants whose seizures had ceased (χ2 = 21.8, df = 1, p = 0.000).

9/44 (20%) of infants with normal development prior to seizure onset had normal development at two years old, and 4/44(9%) had severe-profound developmental delay. Just 1/53 (2%) with abnormal development prior to seizure onset (mild language delay in that infant) had normal development at two years, and 38/53 (72%) had severe- profound delay at two years old or death. All infants with non-assessable development prior to seizures due to neonatal seizure onset had severe-profound delay or were deceased by two years old (for severe-profound delay/death by two years old

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present/absent vs development prior to seizures normal/abnormal/not assessable, χ2 = 60.3, df = 2, p = 0.000).

By epileptic syndrome at onset: Development prior to seizures varied across epileptic syndromes (Table 6.8). Pre-seizure development was not assessable in most infants with EIEE, EME, EIMFS and their variants due to neonatal seizure onset. Normal pre- seizure development was seen in most infants with Dravet syndrome -like (6/7) or ‘focal-multi’ (4/5) epilepsies at onset and none (0/3) with an LGS-like epilepsy. Infants with West syndrome and variants and unifocal epilepsies had a more even mix of infants with normal and abnormal pre-seizure development, with normal pre-seizure development seen in 22/52 (42%) and 8/12 (66%) respectively.

Development at two years old or death was severely impaired in all infants with the neonatal and early-infantile onset epileptic syndromes, EIEE, EME and EIMFS and variants. Normal developmental outcome was seen in some infants with West syndrome and variants and non-syndromic focal epilepsies, and in no infant with other epileptic syndromes. 4/52 (8%) of infants with West syndrome and variants had normal developmental outcome.

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Table 6.8 Development by epileptic syndrome prior to epilepsy onset and at two years old

Development pre-seizures Developmental outcome at 2 years old or death

Epileptic syndrome at N Severe- onset Mild-mod global Normal Abnormal N/A Normal profound global Deceased or isolated delay delay

EIEE 8 0 0 8 0 0 4 4

EME 2 0 0 2 0 0 1 1

10 1 5 4 0 0 4 6

203 EIMFS

WS 52 22 30 0 4 29 18 1

DS 7 6 1 0 0 5 1 1

Other GGE 4 1 3 0 0 2 1 1

LGS 3 0 3 0 0 0 3 0

Focal (uni) 12 8 3 1 5 4 2 1

Focal (other) 11 2 7 2 0 2 6 3

Focal (multi) 5 4 1 0 1 3 1 0

Total 114 44 53 17 10 45 41 18 DS = Dravet syndrome, WS = West syndrome

By aetiology: Prior to seizure onset, 21/31 (68%) of infants with brain malformations had normal development. Among this group, 16/19 (84%) with FCDs and TS had normal pre-seizure development, making up 16/44 (36%) of all infants with normal pre- seizure development. 12/14 (86%) infants with acquired structural aetiologies, 10/11 (91%) with chromosomal conditions and 6/6 (100%) infants with metabolic disorders had abnormal or non-assessable development before seizures began (Table 6.9).

Normal development at two years old was seen only in infants with brain malformations (6/31, 19%) or unknown aetiology (4/38, 10%). The proportion of infants with severe- profound developmental delay at two years old or death was lowest in infants with chromosomal abnormalities and malformations (3/9, 33% and 10/31, 32% respectively) and highest in metabolic disorders and acquired structural conditions (6/6, 100% and 10/14, 71% respectively).

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Table 6.9 Development by aetiology prior to seizure onset and at two years old

Development pre-seizures Developmental outcome at 2 years old or death Aetiology N Mild-mod global Severe-profound Normal Abnormal N/A Normal Deceased or isolated delay global delay

Structural – acquired 14 2 12 0 0 4 10 0

Structural - malformative 31 21 7 3 6 15 6 4

Metabolic 6 0 2 4 0 0 5 1

9 1 7 1 0 6 1 2 205 Chromosomal

Single gene (non- malformative, non- 16 6 4 6 0 7 5 4 metabolic)

Unknown 38 14 21 3 4 13 14 7

Total 114 44 53 17 10 45 41 18

6.3.4 Commentary The absence of a detailed developmental assessment using a standardized and validated tool is a limitation of this study. Determination of developmental level relied on parental and clinician report in many cases as not all had a formal developmental assessment. Because of this, some aspects of development were incompletely reported in some infants, and there is potential for inaccuracies in the data due to inaccurate reporting, for example due to only limited developmental assessments being feasible during clinic appointments, or inaccurate parental report. Similarly, it would be useful to undertake serial developmental assessments over time to characterize the changes in developmental trajectory and the relationship of development to seizures. This was not feasible during this study, and therefore a quantitative assessment of the effect of seizures on development is not possible here. Despite these obvious limitations, useful information can be gleaned from the available data.

Given the magnitude of developmental impairments in many infants and the broad categories chosen for reporting on developmental outcomes, the presence of major (severe-profound) or less major (mild-moderate) delays can be reasonably determined by comparison of reported skills to those expected for age. Finally, a longer period of follow-up will be important in making more nuanced observations and measurements of developmental levels, as more subtle developmental delays and learning difficulties may only become apparent at an older age, as the skills expected for age become more complex. Thus, some infants considered to have normal development at two years old may not ultimately be free of developmental impairments. Conversely, ‘catch-up’ development was observed after two years old in some, such as those who had seizure resolution after epilepsy surgery, such that development in a small number infants was later considered normal (at a comprehensive developmental assessment in some) despite being abnormal at two years old. Detailed developmental assessment of all infants in this cohort at an older age would be informative to precisely assess the presence and magnitude of developmental and learning impairments.

The developmental outcomes in infants with SEI highlight the extreme severity of these conditions. Most infants in this study had developmental impairments at two years old or prior to death, being severe-profound (or non-assessable) in approximately half.

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These infants had a skill level lower than that of a nine-month old when two years old (developmental quotient <35), with the skill level in many considerably lower, as evidenced by a significant number of infants who were not able to babble, had poor head control and were visually inattentive. Previous studies have reported a developmental quotient <70 in 54-78% of infants with epilepsy in the first year of life (Chevrie & Aicardi, 1978; Czochanska et al., 1994; E. Gaily et al., 2016; Matsumoto et al., 1983). The higher rate seen in our study may be due to exclusion of some infants with milder epilepsies and inclusion of those with seizure onset neonatally. An intellectual quotient of <80 was reported in 87% of infants with pharmacoresistant epilepsy in the first year of life (Berg et al., 2012). That study population is more comparable to this study, and the proportion with developmental impairments is similar.

Development was abnormal or non-assessable in approximately 60% prior to seizure onset, suggesting that the underlying aetiology impairs development independently of seizures in many, although the relative contribution of each is not clear. All but one infant with normal development at two years had normal development prior to seizure onset. Thus, the likelihood of a normal developmental outcome where development is already abnormal at seizure onset appears to be very low. While there is a higher likelihood of normal development if the pre-seizure development was normal, the overall rate of impaired developmental outcome in these infants remained very high.

The magnitude of the effect of seizures on development in infants with SEI is not clear. Higher rates of impaired developmental outcome with a younger age of seizure onset (universal in infants with onset under one month old, lower among infants with onset later in infancy) and in those with ongoing seizures at two years old or death may suggest that seizures are impacting development, but may also reflect more severe underlying aetiologies. It is likely that both factors contribute. A clearer suggestion of seizure impact is that many infants were reported to have a developmental slowing or plateau with seizure exacerbation and acceleration after seizure cessation, although this was not able to be quantified here. This phenomenon has been previously reported , particularly in infants with seizure resolution after epilepsy surgery, and improvement appears to be of greater magnitude in infants who have surgery under one year of age (Loddenkemper et al., 2007). Similarly, it has been previously highlighted that

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uncontrolled infant-onset epilepsies are associated with a progressive decline in developmental quotient over time (Berg et al., 2012). These observations highlight the importance of optimizing seizure treatment to minimize seizure-related developmental effects. The observation that the rates of severe-profound developmental delay are higher in those with prompt presentation than those with delayed presentation initially seems at odds with the above. However, this apparent discrepancy may simply reflect the severity of the epileptic syndrome, as most infants with the neonatal and early infantile epileptic syndromes have focal seizures, these typically prompting medical presentation within hours or days of onset.

Development was universally impaired in most epileptic syndromes, with normal development at two years old seen only in some infants with West syndrome and variants, and non-syndromic focal epilepsies. Just 8% of infants with West syndrome and variants had normal development at two years old, this being lower than a previous report of normal outcome in 16% with a median follow-up of 31 months (Hrachovy & Frost, 2003). Notable is the aforementioned ‘catch-up’ development after two years old in a small number of infants in this study, meaning the number of infants with normal development may be higher after a longer period of follow-up. Previous studies have reported abnormal development prior to seizure onset in 68-85% of infants with spasms (Riikonen, 1982); in this study the figure was 42%. The lower figure in this study may reflect differences in the way development was measured, or variability in underlying aetiologies given the relatively small numbers. Nevertheless, that a relatively large number of infants with spasms have pre-existing developmental delay suggests an impact of the underlying aetiology and presumably a lower likelihood of a normal outcome. This has implications for development of novel therapies, as it is possible that treatments that target the underlying aetiology rather than (or in addition to) the seizures specifically may be of greater benefit. Strategies for prevention or early detection of seizures that have been shown to improve development in TS, including serial EEGs in infancy and AED treatment prior to seizures developing if the EEG becomes abnormal (Jozwiak et al., 2011), should be considered in children with other aetiologies (known prior to epilepsy onset) that have a high risk of developing epilepsy. In all infants, early diagnosis and (evidenced-based where available) treatment is a priority.

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In this study, normal developmental outcomes were seen only in infants with focal (FCD, PMG) or multifocal (TS) brain malformations, or unknown causes, with some of the latter suspected to harbour an occult FCD. This finding is important for prognostication, and suggests that aggressive treatment of seizures (especially spasms) is warranted in these infants, including consideration of epilepsy surgery, as the developmental outcome may be normal if detrimental effects of the epilepsy can be avoided, as is shown in the pre-treatment of infants with TS (Jozwiak et al., 2011).

Development prior to seizure onset was normal in some infants in all aetiologic groups other than those with metabolic disorders. Infants with brain malformations had the largest number and proportion of normally developing infants. Given the treatment implications, infants with normal development prior to seizure onset should be strongly suspected of having a brain malformation (and particularly an occult FCD where initial imaging is normal) until proven otherwise (by identification of an alternative aetiology, or the presence of non-congruent clinical features).

Only one infant in this study was diagnosed with an autism spectrum disorder by two years old, this low number reflecting the typically low rate of diagnosis of autism by this age. A number of other infants were documented to have autistic features without a formal autism diagnosis, and the diagnosis was made in some after two years old. Prior studies have reported autism spectrum disorders in 17-36% with infantile spasms and 7% with other seizures in the first year of life (Berg et al., 2011; Saemundsen, Ludvigsson, Hilmarsdottir, et al., 2007; Saemundsen, Ludvigsson, & Rafnsson, 2007). A longer duration of follow-up would be required to determine if the number of infants with autism is similar in this study. The low number of autism diagnoses may also reflect that determination of autistic features is not possible in infants with severe- profound developmental delay who are too impaired developmentally to allow assessment of face regard, social development and repetitive behaviours.

6.4 Comorbidities

One or more neurologic comorbidities was identified in 98 (86%) infants. 72 had a normal head size, 30 microcephaly (nine congenital, 21 acquired), nine macrocephaly (two congenital, seven acquired) and three an unknown head size.

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43 infants had normal tone, 32 had hypotonia, 29 had spastic quadriparesis (including asymmetric bilateral) and four had fluctuating or intermittent appendicular hypertonia. Two infants who were deceased in the neonatal period were hypertonic from birth. Three infants had neonatal hypertonia/rigidity that evolved to hypotonia after commencement of AEDs, later further evolving to spastic quadriparesis. Twelve had a persistent hemiparesis (with either normal contralateral tone or coexisting hypotonia) (8R, 4L), two a transient hemiparesis (2L, 0R) at the age at which seizures were present, and six had an early hand preference (4L, 2R) reported without demonstrable asymmetry on neurologic examination. Tone was unknown in one.

A movement disorder was reported in 32, being of mixed type in 22, dystonia in five, stereotypies in four and unknown type in one. There was no movement disorder in 77, and it was not known whether a movement disorder was present in five. Other neurologic comorbidities reported included strabismus in 19, excess startle in 12, extreme irritability in 11, autonomic instability in 11, hearing impairment in 11 (sensorineural in three, mixed in two, conductive in six) and suspected or proven neuromuscular disorders in six.

Non-neurologic comorbidities presumed to be associated with the underlying aetiology were identified in 43 infants, and included malformations of other organs, skeletal and cutaneous abnormalities, disordered linear growth and dysmorphic features. 47 infants had comorbidities presumed to be complications of a severe neurologic disorder, including lower respiratory tract infections, gastro-oesophageal reflux, constipation and failure to thrive.

A number of infants had comorbidities presumed unrelated to the cause or complications of their epilepsy, such as eczema and asthma. 34 infants required supplemental feeding (excluding those in which a brief period of supplemental feeding was required while hospitalized for an intercurrent illness or seizure exacerbation), delivered by nasogastric or nasojejunal tube in 18 and percutaneous endoscopic gastrostomy or jejunostomy in 16.

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6.4.1 Commentary The majority of infants with SEI have at least one comorbidity (other than seizures and developmental delay), whether neurologic or non-neurologic, related to the underlying aetiology or a complication of severe neurologic impairment. These complications were significantly functionally impairing in many, often required additional treatment or hospitalization, and were life-limiting in some.

The rates of most comorbidities and complications have not been previously reported, although they have been noted to occur frequently. Microcephaly is one of the few comorbidities for which a rate has been reported, occurring in 8% of survivors in a cohort with epilepsy onset in the first year of life (Czochanska et al., 1994). In our study, 26% of survivors had microcephaly, this higher figure presumably reflecting inclusion of those with neonatal onset seizure and exclusion of infants with milder epilepsies.

Some complications provided clues to the underlying aetiology, such as the presence of a hemiparesis or early hand preference in some infants with a focal brain malformation, particular movement disorders in some ‘single gene’ aetiologies and non-neurologic features such as involvement of other organs or cutaneous manifestations. Others were predictable from the underlying disorder or the severity of neurologic impairment and, in some cases, could be improved by early treatment, highlighting the need for surveillance for early detection of potentially treatable complications such as dysphagia, aspiration pneumonia, kyphoscoliosis and urinary incontinence in Dravet syndrome (Catarino et al., 2011).

6.5 Survival

23 (20%) infants were deceased. 18 (16%) infants were deceased by two years old, five of these in the neonatal period.

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Causes of death in the infants deceased before two years old were lower respiratory tract infection in seven12, cardiorespiratory failure in seven, necrotizing enterocolitis in two and unknown in two. The causes of death in infants deceased after two years old were lower respiratory tract infection in four, and one was found dead in bed.

All infants deceased by two years old had severe-profound developmental delay (13) or unknown development due to death in the neonatal period (5). All deceased infants had abnormal or non-assessable development prior to seizure onset. 17/18 had ongoing seizures up to their deaths. 4/5 infants deceased after two years old had severe-profound developmental delay at two years old. The other infant, who had SUDEP, had mild- moderate delay in the context of Dravet syndrome.

Mortality was higher in the infants with younger age of epilepsy onset. 9/17 (53%) infants with neonatal seizure onset were deceased by two years old. A further 2 infants with neonatal onset died after two years old (total to the time of writing 11/17, 65%). 5/21 (24%) infants with seizure onset between 1-3 months died before age two years, 2/33 (6%) infants whose seizures began between 3-6 months (with one more after two years, total 3/33, 9%) and 3/31 (10%) with seizure onset between 6-12 months old (χ2 = 23, df = 4, p = <0.001). Two infants with seizure onset between 12-18 months died after two years old, but all were alive at age two years.

Mortality to two years old was very high in the neonatal and early infantile-onset syndromes (EIEE 4/8, EME 1/2 and EIMFS 6/10) and lower in other syndromes, including 1/52 (2%) in those with West syndrome and 1/12 with unifocal epilepsies. 1/7 infants with Dravet syndrome / Dravet syndrome -like epilepsies died before two years old, and another 1 after two years old.

At least one infant in all aetiologic categories was deceased, with the exception of those with acquired structural conditions. 4/16 (25%) with single gene disorders (SCN2A 2, SCN8A 1, TBC1D24 1), 2/9 (22%) with chromosomal abnormalities, 7/38 (18%) with

12 One infant was found dead in bed, and was thought by her clinicians to have had SUDEP, but formal cause of death at autopsy was listed as lower respiratory tract infection.

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unknown aetiologies, 1/6 (16%) with metabolic disorders and 4/31 (13%) with brain malformations (all having either a diffuse/complex malformation and/or other medical conditions) were deceased.

6.5.1 Commentary The mortality rate in SEI is high, with one in six deceased by two years old and 20% at the time of writing (surviving infants aged 2.7-5.6 years). Prior studies of survival in hospital-based cohorts of infants with epilepsy onset in the first year of life reported mortality of 11-15% in infants followed for 1-6 years (Chevrie & Aicardi, 1978; Czochanska et al., 1994; Matsumoto et al., 1983). The higher rate seen here probably reflects inclusion of neonates in this study.

The proportion of infants with neonatal onset of seizures and/or one of the neonatal/early infantile-onset epileptic syndromes who are deceased was extremely high. All infants deceased before two years old had severe-profound (or non-assessable) developmental delay, with approximately 3/10 severely delayed infants deceased by two years old.

High mortality in some genetic aetiologies has been previously reported. The mortality rate in Dravet syndrome is reported to be 3.75-17.5% (Bureau et al.; Sakauchi et al., 2011; Skluzacek et al., 2011). A recent study reported a mortality rate of 15.84/1000 person years, and noted that this equates to approximately 15% mortality by 10 years after diagnosis (Cooper et al., 2016). In our study, 2/7 (28%) of infants with Dravet syndrome / Dravet syndrome -like epilepsies were deceased at the time of writing. It is not clear whether the higher rate in our study is statistically significant given the small group, this finding does raise the possibility that mortality rates in Dravet syndrome may have been underestimated. Infants dying under two years of age may have escaped diagnosis unless the epileptic syndrome was recognised and the aetiology actively pursued. This will become clearer with increasing awareness of this diagnosis from the time of initial presentation and increased availability of molecular testing.

Early deaths have been reported in some infants with SCN2A and SCN8A mutations (Howell et al., 2015; Larsen et al., 2015; Veeramah et al., 2012). In this study, 3/4

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infants with a mutation in either of these genes were deceased by two years old. While the low numbers in this study mean that accurate mortality rates cannot be determined for individual aetiologies, the findings of this study are consistent with previous reports. Infants with sodium channelopathies should be considered to have a high risk of death. Particularly with Dravet syndrome, the underlying aetiology likely confers a risk over and above that of other infants with SEI with mild-moderate developmental delay and seizure onset outside the neonatal period. The mechanism(s) for SUDEP in these disorders is not clear. Mouse models of Dravet syndrome and SCN8A encephalopathy report a terminal tonic-clonic seizure precedes the SUDEP. Secondary generalized tonic-clonic seizures preceding SUDEP were also observed in adult patients, suggesting that mechanisms for SUDEP in sodium channelopathies may be similar to those in other epilepsies (Kalume et al., 2013; Ryvlin et al., 2013; Wagnon et al., 2015)

Approximately one in five infants with an unknown aetiology was deceased by two years old. This figure is particularly important in the context of reproductive counselling implications for the family. Without knowing the aetiology, accurate reproductive counselling cannot be provided and most reproductive interventions to prevent a second affected child cannot be used. This is a major issue with early-life conditions such as SEI, as parents are often young and want more children. Finding the cause of the epilepsy in infants with unknown aetiology will be of significant benefit to such families.

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Chapter 7: Diagnostic investigation

7.1 Timing, method and yield of diagnosis

The timing and method of aetiologic diagnosis are listed in Table 7.1 and further discussed below. Further detail on individual patients is provided in Appendix I.

Table 7.1 Timing and method of SEI diagnosis in 114 infants

Timing/method of diagnosis* Number Aetiologies

Clinical diagnosis prior to seizure onset 28

Prenatal diagnosis 4 Antenatal ultrasound 4 Tuberous sclerosis 3, achondroplasia 1

Postnatal diagnosis 23

Clinical features + brain imaging +/- tests 14 Neonatal HIE 4, periventricular leukomalacia 3, of blood and CSF complicated meningitis 2, neonatal HIE and hypoglycaemia 2, perinatal stroke 2, neonatal ischaemic injury (mechanism unknown) 1 Chromosomal testing$ 5 Trisomy 21 4, isodicentric chromosome 15 1

MRI brain 2 Aicardi syndrome 1, malformation of cortical development (other) 1 White cell enzyme testing 1 Tay-Sachs disease 1

Single gene testing# 1 Mitochondrial disorder 1

Unknown if pre- or postnatal diagnosis 1 Trisomy 21 1

Clinical diagnosis after seizure onset 33

Clinical assessment and Tier 1 testing 21 Clinical features 4 SCN1A mutation 2*, SCN8A mutation 1*, Sotos syndrome 1* MRI brain 13 Focal cortical dysplasia 3, polymicrogyria 3, lissencephaly 2, malformation of cortical development (other) 1, pontocerebellar hypoplasia 1, Sturge-Weber syndrome 1, tuberous sclerosis 1, Aicardi-Goutières syndrome 1* Chromosomal microarray 4^ Chromosome 2q24.3 deletion 1, Wolf-Hirschhorn syndrome 1, Chromosome 15q21.3q22.2 deletion, Chromosome 17p13.3 deletion^ Urine metabolic screen 1 Molybdenum cofactor deficiency 1

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Timing/method of diagnosis* Number Aetiologies

Tier 2 testing 3 Mitochondrial disorder 2, PNPO deficiency 1*

Repeat MRI brain 9 Focal cortical dysplasia 7&, tuberous sclerosis 1, malformation of cortical development (other) Tier 3 testing 0

Diagnosis after seizure onset (research) 15

MRI brain 4 Focal cortical dysplasia 4

Genetic testing – single gene 1 TBC1D24 mutation

Genetic testing – WES, MIPS or WGS 10 KCNQ2 mutation 3, SCN2A mutation 2, KCNT1 mutation 1, SCN1A mutation 1, SCN8A mutation 1, SMC1A mutation 1, SYNGAP1 mutation 1

No diagnosis 38

Total 114 $Banded karyotype, FISH or chromosomal microarray

#In the context of family history of genetically confirmed mitochondrial disorder in similarly affected sibling *Suspected diagnosis which resulted in targeted single or multigene testing to confirm diagnosis

& 1/7 was a suspected diagnosis which was confirmed with additional testing

^Diagnosis also made on MRI brain (first scan)

7.1.1 Diagnosis prior to seizure onset 28 (25%) infants had an aetiologic diagnosis made prior to seizure onset. This number included all 14 infants with an acquired cause. Nine infants had diagnoses made on the basis of non-neurologic clinical and/or imaging features (trisomy 21 5, TS 3, achondroplasia 1), four through investigation of developmental delay or regression (Aicardi syndrome in 1, isodicentric chromosome15 in 1, malformation of cortical development (other) in 1, Tay-Sachs disease in 1) and one because of developmental delay and a family history of similar (mitochondrial disorder in 1).

Four infants had a diagnosis made on antenatal ultrasound (3 TS, 1 achondroplasia). 23 infants had a diagnosis made postnatally. Diagnosis was made in the 14 infants with acquired aetiologies with a combination of clinical features, brain imaging and, in some cases blood tests (e.g. blood glucose level) and cerebrospinal fluid (e.g. cell count and gram stain/culture/PCR) analysis. Five infants had a diagnosis made on chromosomal

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testing (4 trisomy 21, 1 isodicentric chromosome 15), two on MRI, one on white cell enzyme analysis and one on single gene testing (in the context of known genetic disorder in other family members). The timing of diagnosis was not known in one infant with trisomy 21.

Eleven of 14 (79%) infants with a non-acquired aetiology had the genetic basis confirmed.

7.1.2 Diagnosis after seizure onset: standard clinical testing 33/86 (38%) infants with no aetiologic diagnosis at seizure onset (33/114, 29% of all infants) had a diagnosis made with standard clinical investigations (26) or suggested with standard clinical assessment +/- investigations and then confirmed with targeted testing (7). MRI brain imaging made or suggested the diagnosis in 22/33 (67%); these diagnoses were made by the reporting radiologist or upon multidisciplinary clinical review of this imaging, for example at our epilepsy case conference in concert with the EEG and clinical features of the patient’s epilepsy. Infants with diagnoses suspected clinically and confirmed with targeted testing were two with Dravet syndrome who had SCN1A mutations identified on single gene testing, one with a phenytoin-responsive EIMFS-like epilepsy and movement disorder who had an SCN8A mutation identified on a clinical multigene panel, one with a clinical diagnosis of Sotos syndrome who had an NSD1 mutation identified on single gene testing, one with brain imaging suggestive of Aicardi Goutières syndrome and a family history of a similar presentation in a sibling who had an RNASEH2B mutation identified on a gene panel, one whose seizures were responsive to pyridoxal-5-phosphate who had urine and CSF amino and organic acid profiles consistent with those seen in PNPO deficiency, and an infant with an FCD suggested clinically and on repeat MRI brain imaging that was later confirmed on histopathology of brain tissue taken at epilepsy surgery. The genetic basis of the identified aetiology was confirmed in 14/33 (42%) infants.

7.1.3 Diagnosis after seizure onset: research imaging review Four infants had a diagnosis made on research review of MRI brain imaging.

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FCDs were identified in four infants whose brain imaging had been reported as normal. In three, the dysplasia was FCD visible on the original scan(s). In one, the initial imaging showed only some subtle asymmetries; repeat imaging was performed and confirmed an FCD.

Table 7.2 Occult focal cortical dysplasia identified on research imaging review

A) Axial T2-weighted imaging in infant IEE12007 at four months old showing a an area of T2- hypointensity in the right temporal lobe suspicious for a focal cortical dysplasia (arrow)

B) Coronal T2-weighted imaging in infant IEE12007at two years old showing blurring of the grey- white junction in the right temporal lobe, consistent with a focal cortical dysplasia (arrow)

C) Axial T2-weighted imaging in infant IEE12007 showing blurring of the grey-white junction in the right temporal lobe, consistent with a focal cortical dysplasia (arrow)

All four infants were suspected of having a FCD after my clinical assessment for this study. Suggestive features included a hemiparesis in one, spasms in three (being asymmetric in two and late-onset in one), (uni)focal seizures in two, and a focal interictal epileptiform EEG in all four. A genetic basis was identified in two of these infants (DEPDC5, BRAF), a VOUS found in one (MTOR) and no variants of interest were identified in one.

FCD is suspected in a further six infants who underwent a study clinical assessment; three had findings suggestive but not diagnostic of FCD identified on research imaging review. No further imaging was performed in these six infants because they were seizure free.

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7.1.4 Diagnosis after seizure onset: research genetic testing An aetiologic diagnosis was made on research genetic testing in 11 infants.

One infant had a diagnosis made on targeted single gene testing (TBC1D24), three on MIPS gene panel (KCNT1 1, SCN1A 1, SCN2A 1), six on WES gene panel (KCNQ2 3, SCN2A 1, SCN8A 1, SMC1A 1) and one on WGS (SYNGAP1).

One infant had a dual diagnosis made. Neurocutaneous melanosis was diagnosed clinically (based on clinical features and brain imaging), but there was doubt that this was the cause of her SEI as her epilepsy was much more severe than previously reported in this condition. For this reason, this infant underwent research genetic testing, which identified a KCNQ2 mutation, which is entirely consistent with her epilepsy phenotype (Weckhuysen et al., 2013; Weckhuysen et al., 2012).

11 infants with a malformative or metabolic aetiology had research genetic testing through this or other studies. Two infants with malformative causes (one whose malformation was identified on research clinical review) had pathogenic mutations in NPRL3 and DEPDC5 and one with a metabolic aetiology had a possibly pathogenic mutation in FARS2 (detail of assessment of pathogenicity provided in Aetiologies chapter).

7.1.5 Yield of diagnostic investigations Current standard clinical investigations including research imaging review: All 86 infants with no diagnosis known prior to seizure onset had investigation of the aetiology of their SEI, although there was variability in the type and number of tests done and the order in which they were performed. Table 7.3 lists all investigations performed and calculates their yield. Please refer to section 3.5.4 for details of the metabolic urine, blood and CSF tests performed in each tier. Table 7.4 considers the yield of each tier if it were only performed in infants who did not have a diagnosis made with the prior tier, this being a closer approximation of the yield within the current ‘tiered’ diagnostic pathway. These tables include infants whose diagnosis was made clinically prior to research review or on research imaging review.

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Table 7.3 Clinical investigations and research imaging review in 86 infants with unknown cause at the time of presentation

Tier Investigation Infants in whom Infants with diagnostic Yield investigation or suggestive finding (n) (%) performed (n) 1 MRI 85 13#$ 15% Chromosomal 74 4* 5% microarray Metabolic urine tests 74 1& 1% Metabolic blood tests 82 0^ 0% Suggested clinically 4% (all four confirmed with AND other Tier 1 tests genetic testing) did not identify aetiology TOTAL 21 2 Metabolic and 55 0 0% chromosomal blood tests Metabolic CSF tests 61 3 5% TOTAL 3 Repeat MRI 39 9@ 23% TOTAL 9 3 Liver, muscle and skin 8 0 0 biopsy TOTAL 0

Research MRI 81! 4 imaging review TOTAL 4

#12 diagnostic, 1 suggestive of Aicardi-Goutières (later genetically confirmed)

$ 1 had a dual diagnosis identified (neurocutaneous melanosis, not thought to be the cause of epilepsy)

*1 had diagnosis also made on MRI-B (lissencephaly, associated with chromosome 17p13.3 deletion)

&1 had urine metabolic screen consistent with molybdenum cofactor deficiency

^2 had elevated serum lactates which were not considered ‘significant’ in and of themselves despite ultimate mitochondrial diagnoses (but elevated CSF lactates in these 2 patients were subsequently considered significant) as a number of patients had elevated serum lactates, usually transiently, and ultimately had a non- mitochondrial disorder identified or no other evidence to support a mitochondrial disorder.

%4 had clinical diagnoses suggested and later genetically confirmed on clinical testing– 2 Dravet syndrome (SCN1A), 1 Sotos syndrome (NSD1), 1 phenytoin-responsive epilepsy and movement disorder (SCN8A)

@7 diagnostic, 1 suggestive of FCD (later further suggested by PET and confirmed by histopathology at epilepsy surgery)

!MRI not performed in 1, and not available for review in 4. 4/5 infants had a known diagnosis (lissencephaly 1, molybdenum cofactor deficiency, mitochondrial disorder, SYNGAP1 mutation).

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MRI brain was the investigation with the highest diagnostic yield. 85/86 infants had at least one MRI, 26/85 (31%) having a significant result on any MRI. MRI was diagnostic in 24 infants, the diagnosis made on the first MRI in 12, repeat MRI in eight and research review of MRI (+/- repeat MRI) in four. Two had findings suggestive of a particular aetiology, both confirmed with targeted tests (1 Aicardi Goutières syndrome – genetic testing, 1 FCD – PET and histopathology). Research imaging review of the remaining 58 infants identified three infants with imaging features that were suggestive but not diagnostic of FCD (and no additional investigation was performed to further investigate that possibility), one with a diagnosis made that was not thought to be cause of her epilepsy (neurocutaneous melanosis in an infant who was later found to have a KCNQ2 mutation, her phenotype being consistent with the latter), 34 with non-specific findings, 18 with normal imaging and three with unknown imaging findings (two of whom had a known metabolic diagnosis).

CMA identified an aetiology for SEI in 4/74 (5%) infants, one of whom also had a diagnosis made on initial MRI. Ten (14%) infants had a CNV not thought to be the cause of their SEI. In nine infants, the CNV was inherited from an unaffected (or not similarly affected – one was inherited from a mother with mild intellectual disability but not epilepsy13) parent. Parental testing was not performed in one, although the variant itself was thought unlikely pathogenic (a novel 7p22.1 duplication containing no genes known to be associated with epilepsy).

Metabolic testing was of low yield, with one diagnosis made or suggested on Tier 1, three on Tier 2 and none on Tier 3. Three of four infants with a metabolic diagnosis had neonatal onset of seizures, and the two with mitochondrial disorders had elevated serum and CSF lactate. Non-specific abnormalities in urine amino acids and organic acids and CSF amino acids were seen in a number of infants, attributable to factors other than

13 Although it is recognised that CNVs can have variable neurologic phenotypes (and thus be plausible that a CNV could produce intellectual disability in one family member and epilepsy and developmental delay in another), this CNV was not thought to be the cause of this infant’s epilepsy for two reasons. Firstly, this CNV contained no known epilepsy genes. Secondly, this infant’s phenotype was typical of KCNQ2 encephalopathy, and a missense mutation that has been previously reported to be pathogenic was identified.

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aetiology (e.g. intake of particular diets and medication), and did not prompt additional investigations. A number of infants had elevated serum lactates which were attributed to difficult blood taking, repeat testing being within normal limits. All infants who underwent Tier 3 testing to investigate for mitochondrial disorders did not have elevated CSF lactate, nor persistently elevated serum lactate; none of these infants had evidence of respiratory chain enzyme dysfunction or mitochondrial depletion on Tier 3 testing.

Table 7.4 Yield of each tier of investigations in infants with unknown cause at the time of presentation

Investigation(s) Infants with no Some/all of tier Diagnosis made No diagnosis known aetiology done (n) (yield (%)) made prior to each tier (n)

Tier 1 86 86 (100%) 21 (24) 65

Tier 2 65 53(82%) 3 (6) 62 (50)

Repeat MRI 62 37 (60%) 8 (22) 54 (29)

Tier 3 54 7* (13%) 0 (0) 54 (8)

Research imaging N/A 81 (94%) 4 (N/A) N/A review *The 8th infant to have biopsies (as noted in earlier table) had a diagnosis made on a Tier 1 test (chromosomal microarray). All testing was done in quick succession in this infant because of (expected) neonatal death.

Research genetic testing: 44/49 (90%) infants with unknown aetiology after clinical investigation and research imaging review underwent research genetic testing. The remaining five did not consent to research genetic testing. The aetiology was identified in 11/44 (25%) (Table 7.5).

Just one infant, who had DOORS syndrome, had targeted single gene testing that which confirmed the suspected TBC1D24 mutation (Campeau et al., 2014). 10/43 (23%) infants who had multigene testing had a cause identified. WES results are pending for 5/43 infants. 30 infants had >1 type of research genetic testing performed. Three infants with a diagnosis made on WES had had a negative MIPS gene panel that included the causative gene (KCNQ2 in 2, SCN2A in 1); the reasons for these false negative MIPS results are likely related to technical issues associated with the multi-inversion probes.

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The SYNGAP1 mutation was not identified on WES as the reference genome to which the data was mapped gave a mapping quality of zero for most SYNGAP1 reads. The mutation was identified on WGS; when WES data was reviewed subsequent to that, it was determined that the mutation could have been identified on WES were a different version of the reference genome (which does map SYNGAP1 reads) used.

Variants of uncertain significance were identified in 13 (30%). These will require additional evidence (e.g. additional cases, functional data) to determine pathogenicity.

No ‘incidental’ findings were made. Genes that cause disorders those than epilepsy, such as breast cancer due to mutations in the BRCA1 gene, that the American College of Medical Genetics and Genomics recommends reporting of incidental findings, were not analysed (Kalia et al., 2016).

Table 7.5 Research genetic testing performed on infants with unknown aetiology after clinical investigation and research imaging review

Investigation Infants in Aetiology Variant of No Other whom determined unknown aetiology investigation significance identified performed (n) Single gene testing

TBC1D24 1 1 0 0 0 sequencing*

Multigene testing

MIPS gene 32 3 0 19 10 panel inadequate coverage

WES 40 6 13 17 5 pending WGS 1 1 0 0 0

*Infant had DOORS syndrome diagnosed on research clinical assessment which led to targeted testing

7.1.6 Commentary In this study, the diagnostic yield increased from 53% to 67% through WES and review of brain imaging. A further 7% have a specific diagnosis suspected from research

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clinical and imaging review, but additional testing has not been performed (or is pending) to confirm these suspected diagnoses.

The discussion below will be confined to infants with no diagnosis at epilepsy onset, as those with a known diagnosis were not further investigated.

All but one infant had an MRI brain; this infant had a similarly affected older sibling who had normal imaging. Like previous studies, MRI brain imaging was the highest yield diagnostic investigation, with 31 % of those with an unknown aetiology at epilepsy onset having a diagnostic (or highly suggestive) MRI. This figure is at the lower end of 25-56% yields reported in other studies (Berg et al., 2009; Eltze et al., 2013; Osborne et al., 2010; Poulat et al., 2014; Wirrell et al., 2015) , although some of these studies included infants whose diagnosis was made prior to epilepsy onset, and had higher rates of acquired aetiologies, both of which increase the yield of imaging. This study demonstrated that the yield of MRI increased with detailed imaging review and/or repeat imaging and/or additional information from epilepsy characterization. Imaging may ultimately yield additional diagnoses - if the six infants with suspected FCDs had these confirmed, the yield of imaging would increase to 36%, and additional diagnoses in other infants may be made at an older age when subtle FCDs are more easily detected. The proportion of infants with infantile spasms and (unifocal) seizures who have an FCD is particularly high; this is discussed in Chapter 6 (Clinical features). MRI brain imaging is clearly a critical investigation in SEI, and the importance of detailed imaging review by a neuroradiologist with a knowledge of the clinical findings (e.g. clinical and EEG localization of seizures) cannot be underestimated.

CMA was diagnostic in 5%, in line with previous studies (Allen et al., 2015; Mefford et al., 2011). An important consideration with this testing is the identification of CNVs of unknown significance in 14%. After parental testing, all of these were ultimately considered non-causative. The relatively low, but still significant diagnostic yield, should prompt consideration about whether CMA should be performed either in a targeted group of infants only, or as a second (or later) tier test rather than as first-line.

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Metabolic testing in this study was performed in a tiered approach, with relatively non- invasive and cheaper tests performed first, and treatable disorders tested for across tiers one and two. Despite the extensive testing performed, just four had a metabolic diagnosis made, only one of which was treatable. A diagnosis or likely diagnosis would have been made with WES gene panel testing on three infants, including the infants with a treatable disorder. The yield of metabolic testing in this study is similar to other recent studies of infantile spasms or severe epilepsies in infancy and childhood, which reported yields of 4.5% and 7% (Mercimek-Mahmutoglu et al., 2015; Wirrell et al., 2015). Given this, consideration needs to be given to changing the current protocol for metabolic testing; this will be considered further in the Health Economic section below.

To my knowledge, this is the first population-based study to utilize NGS in epilepsy. In this study genetic testing made an aetiologic diagnosis in 15 infants (4 on clinical testing, 11 on research testing)whose diagnosis would not be made on standard imaging, metabolic and chromosomal testing. Prior non-population based studies of NGS (using gene panels or unbiased WES) in populations with severe epilepsies (only those with >10 patients considered) have yielded a diagnosis in 10-48% (Allen et al., 2016; Carvill, Heavin, et al., 2013; Consortium et al., 2013; Gokben et al., 2016; Lemke et al., 2012; Mercimek-Mahmutoglu et al., 2015). The significant variation in yield is likely accounted for by the phenotypes of the included infants (e.g. some studies included infants with Dravet syndrome who have a very high likelihood of having a mutation identified), and whether prior genetic testing had been performed. This study shows that, at a population level, the yield of multigene testing (performed as the first non- chromosomal genetic test) is at least 15/53 (28%) (49 with research genetic testing, 4 with diagnosis made on clinical genetic testing), which is comparable to the yield of brain imaging, and would argue for its inclusion early in the diagnostic pathway. This proportion is likely to increase following completion of WES gene panel in those in whom a result is pending, and clarification of the significance of VOUS. Moreover, genes currently unidentified will add to the pool to be analysed in future WES studies, and potentially increase yield.

The overall use of multigene testing is broader than that above. As noted earlier, the WES gene panel would have also made the diagnosis in three infants with metabolic

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disorders, and determined the genetic basis in a number of infants with structural- malformative conditions, thereby providing additional benefit in potentially replacing current diagnostic tests (metabolic testing) and providing information to allow accurate reproductive counselling where an aetiologic diagnosis was made using another technique. The proportion of infants with malformative diagnoses in whom an underlying basis could be determined with WES performed on DNA from blood or saliva is not known as not all infants with malformations had genetic testing.

The genetic basis of SEI is highly heterogeneous, with significant phenotypic variability and genotypic heterogeneity (rev in (McTague et al., 2016)). Previous studies have suggested that multigene testing is better than targeted single gene testing for patients with epilepsy. The experience in this study would agree with that assessment. Less than half of the infants with a single gene disorder diagnosed had a single gene strongly suspected (either by the clinician or by me on research review) early in the course of their epilepsy (a number had a small set of genes thought likely early, or had a specific cause deemed clinically likely later in their clinical course), and no individual genetic aetiology was seen in more than three individuals.

In this study, the characteristics of the mutations identified on single gene testing and MIPS gene panel were such that it would be expected they would also be identified on a WES gene panel. In contrast, three infants had diagnoses made on WES gene panel that were not detected on MIPS gene panel (which included the causative genes). The specific reasons for this discrepancy may be attributed to the relative strengths and weaknesses of each technique. Testing of every infant with both techniques was not performed to assess the differences in the performance of both at the group level. While MIPS testing is considerably cheaper (estimated at $1 per gene (O'Roak et al., 2012)), the advantage of a WES-based gene panel is that one can later use this data to analyse the exome in a non-biased manner without the need for additional sequencing if the gene panel is negative or as new genes are discovered. WES-based gene panels (and MIPS gene panels) are limited in the types of mutations they detect. Specifically, exonic variants in poorly covered genes, non-exonic variants, some larger genomic variants, and other variants such as triplet repeats will not be identified. Some of the limitations to WES-based gene panels may be overcome by the use of WGS, but this technique

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remains predominantly in the research domain due to factors of both cost and the challenges of variant interpretation. Such testing, once available, will provide a diagnosis in some infants whose cause is unknown after WES.

VOUS were identified in 30% of infants undergoing multigene testing. Parental testing of some of these variants is pending. Variants for which parental testing shows a pattern of inheritance potentially consistent with that of the relevant gene (e.g. de novo variant in dominant gene with unaffected parent)will require functional studies or the identification of additional cases to determine pathogenicity. This requires additional resources including large multicentre/international disease databases and collaborations with laboratories undertaking functional work, which can be expensive and time- consuming, not to mention produce uncertainty and anxiety for the family. The introduction of CMA testing saw similar issues (rev in (Hehir-Kwa, Pfundt, Veltman, & de Leeuw, 2013), albeit on a considerably smaller scale. As knowledge and available data has increased, such as with the development of ExAC and recently GNomad which houses over 100,000 exomes and 15,000 genomes from controls (www.gnomad.broadinstitute.org), these issues have reduced. Hopefully, with such coordinated efforts by researchers and clinicians in the genomics field to catalogue and categorise normal and disease-associated variation, the interpretation of NGS will be more straightforward in years to come. A significant issue in the interpretation of variants in SEI is that these epilepsies are highly phenotypically and genotypically heterogeneous. As more cases of each gene are reported, clarification of the phenotypic spectrum and which clinical features are and are not seen with mutations in a particular gene occurs. This is exemplified by the finding that some genes such as SCN1A are only very rarely associated with epileptic spasms (Harkin et al., 2007). Thus, determination of pathogenicity is determined not just by the characteristics of the genetic variant, but also whether the phenotype correlates with that known for the mutated gene; the neurologist therefore plays a critical role in this process. Further clinical studies delineating the phenotypic spectrums for each gene will also be invaluable.

No infant had a significant ‘incidental’ finding on multigene testing. Due to the panel- based analysis, only genes associated with infant seizures were studied, meaning non- epilepsy-associated ‘diagnoses were not possible. This becomes a potential issue if

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unbiased exome analysis is performed; a recent large study of clinical exome identified incidental findings in genes unrelated to the phenotype in 4.6%, with management implications in most(Yang et al., 2014). Reporting and managing incidental findings is a significant issue in the use of NGS, with debate about which findings should be reported, and further study needed to determine the clinical utility of reporting such information. Minimising the number of incidental findings identified is an argument in favour of using a WES-based gene panel, rather than unbiased exome analysis, as the initial NGS technique.

7.2 Implications of making a diagnosis on research imaging or genetic testing

Fifteen infants had a diagnosis made on research imaging or genetic testing that was not made in standard clinical testing. In 2/15, diagnosis was made post-mortem. The diagnosis led to a change in management in one infant with FCD; this infant had epilepsy surgery 16 months ago at 3.5 years old, has been seizure free since and has made some developmental gains. Making a diagnosis influenced the choice of AEDs in infants with an SCN2A mutation, as sodium channel blocking drugs are reported to be more effective than other AEDs (Howell et al., 2015). Prognostic information was able to be provided in a number of cases. For example, seizures often settle by early childhood in those with KCNQ2 mutations (Weckhuysen et al., 2013; Weckhuysen et al., 2012), which will influence decisions about longer term use of AEDs and interpretation of the nature of any new paroxysmal episodes. Accurate reproductive counselling is also available to the families of all infants with genetic diagnoses made on research testing. While recurrence risk is low for most of these families, as the infants had de novo mutations (NB risk is not zero given the possibility of parental gonadal mosaicism), a significant recurrence risk was present in two families (one with mosaicism in an unaffected parent and another in which both parents are heterozygous carriers for a recessive disorder).

7.2.1 Commentary The additional yield provided by NGS in the diagnosis of many disorders is now well known, and the important implications of making a genetic diagnosis have been reported in a number of papers, including one study of Dravet syndrome (Brunklaus et

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al., 2013; Stark et al., 2016). Similar benefits apply to making a genetic diagnosis in infants with SEI at both an individual and a group level. These benefits are difficult to quantify, but are important topics for future study.

At the individual level, despite the fact that few genetic causes of SEI have a specific treatment, the benefits of making a diagnosis are many and their impact on the infant and family should not be underestimated. Making a diagnosis ends the ‘diagnostic odyssey’, providing an explanation for the infant’s condition, and obviating the need for further, often invasive and expensive, investigation. Even in the absence of a treatment of major effect, there are often implications for management. These can include targeting AED treatment – such as preferential use of sodium channel blockers for SCN2A and SCN8A encephalopathy, but avoidance of these same drugs in infants with SCN1A mutations (Guerrini et al., 1998; Howell et al., 2015; Larsen et al., 2015). Later-onset disease manifestations can be screened for and early treatment implemented. Information is available to guide planning for the future, including access to services and, if appropriate, redirection to palliative care. There are significant psychosocial implications for families including laying to rest perceived parental or medical ‘blame’ for causing the infant’s condition and access to disease-specific support groups and conferences.

For a group of extremely severe, genetically heterogeneous conditions with variable recurrence risk such as SEI, a major issue in not making a diagnosis is the inability to provide accurate reproductive counselling to families. For these early onset conditions, parents are often young, and many plan to have more children. Although de novo dominant mutations, with very low recurrence risk, are most common, the number of families in this study with two affected children highlight that there is a significant recurrence risk in some. Making a diagnosis allows accurate counselling about recurrence risk, and the use of preconception or prenatal technologies to avoid or detect a second affected child where this is desired. Despite this major benefit of diagnosis though, there remain issues with accurate counselling in some cases. Firstly, in the case of ‘de novo’ dominant mutations, the determination of wild type state in the parent is made on testing of peripheral (somatic) tissue and does not exclude the possibility of gonadal mosaicism. This is being increasingly reported in the literature (Xu et al.,

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2015), highlighting that the risk of a second affected child in ‘de novo’ dominant scenarios is not zero, as seen in one family in this study. Secondly, mutations in some genes are associated with a very broad spectrum of severity, from ‘benign’ epilepsies to SEI such as SCN1A and SCN2A (Escayg et al., 2000; Harkin et al., 2007; Heron et al., 2002; Howell et al., 2015). For some genes, a genotype-phenotype correlation that predicts the severity of disease is not available, making prediction of prognosis difficult in infants.

In SEI, making a diagnosis on brain imaging is just as important as making a genetic diagnosis because of the treatment implications. Infants with occult FCDs may be candidates for epilepsy surgery, which can be curative, and significantly improve developmental outcomes if performed early.

Diagnosis of both genetic and malformative causes in infants with previously unknown aetiology has implications across the patient group and for health systems, resulting from a better understanding of the causes of this group of disorders. This information will inform changes to diagnostic pathways to improve diagnostic yield in clinical practice, likely involving high quality, MRI brain imaging with epilepsy-specific sequences and imaging review conducted with clinical and EEG findings in mind, and next-generation genetic testing. This will be further studied and discussed in the Health Economics section below, taking into account the cost (in addition to yield) of these diagnostic investigations.

Finally, a better understanding of the spectrum of aetiologies in SEI will guide research priorities in these conditions, providing neurobiologic insights into how these conditions develop, and guiding development of novel aetiology- or pathway-specific treatments.

7.3 Health economics of diagnostic investigation

This section considers the cost and yield of a number of simulated diagnostic pathways at a group level in infants with no aetiologic diagnosis at seizure onset. Pathways without WES and with WES at different points in the pathway were considered, and each scenario modelled twice – with all infants with unknown cause and only with

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infants whose seizures were ongoing. Individual patient data that was used to model each pathway is provided in Appendix M.

7.3.1 Modelled diagnostic pathway Eighty-six infants underwent Tier 1 +/- subsequent testing. The current diagnostic pathway made an aetiologic diagnosis in 38 infants.

Where all infants continued in the diagnostic pathway, the inclusion of WES increased the number of diagnoses by 10. In the final scenario, in which Tier 2 was omitted, two fewer diagnoses were made. These were two infants with mitochondrial disorders, in whom the model assumed a suspected diagnosis would be made on Tier 2 testing, prompting targeted testing. Neither of these infants had a diagnosis made on the WES performed earlier in the scenario. In the research genetic testing these infants had undergone, one had a potentially pathologic homozygous mutation in the FARS2 gene (further detail in the Aetiologies chapter), and the other had had WES data analysed with that of other affected and unaffected family members with no cause found. Both infants had a mitochondrial diagnosis suspected by their clinician after Tier 1 testing. Thus, it is likely this would have been further investigated in clinical practice even if Tier 2 and 3 testing were not part of the standard diagnostic pathway.

Fewer additional diagnoses were made in the scenarios in which investigation ceased if seizures had ceased. One infant whose diagnosis was made on WES and whose seizures ceased between three and six months did not have a diagnosis reached in the diagnostic pathways in which WES was at step 4 or 5. Two infants with diagnoses made on WES had seizures cease between one and three months; no diagnosis was made in these infants in scenarios in which WES was at step 3 or later. Two infants whose diagnoses were made on repeat MRI (1) and WES (1) had seizures cease at <1 month; in these scenarios, they were not investigated beyond Tier 1 and thus did not have a diagnosis made in any scenario.

The number of infants with diagnoses made in each diagnostic pathway modelled is shown in Table 7.6 below.

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Table 7.6 Number of diagnoses made using each diagnostic pathway modelled

Scenario Step 1 Step 2 Step 3 Step 4 Step 5 Diagnoses made if all Diagnoses made if only infants investigated to infants with ongoing diagnosis or pathway seizures continue the complete pathway of investigation 1 Tier 1 Tier 2 Repeat Tier 3 - 39 39 MRI 2 Tier 1 Tier 2 Repeat Tier 3 WES 49 44 MRI 3 Tier 1 Tier 2 Repeat WES Tier 3 49 44 MRI 4 Tier 1 Tier 2 WES Repeat Tier 3 49 45 MRI 5 Tier 1 WES Tier 2 Repeat Tier 3 49 47 MRI

232 6 Tier 1 WES Repeat Tier 2 - 49 47 MRI 7 Tier 1 WES Repeat - - 47 45 MRI

In addition to the number of diagnoses changing with the addition of WES, the number of patients having each tier performed changes with each scenario (Tables 7.7 and 7.8). Figures 7.1-7.4 show the number of tests performed and diagnoses made in a diagnostic pathway that does not include WES and a pathway in which WES is performed after Tier 1 testing.

Scenario Tier 1 Tier 2 Repeat MRI Tier 3 WES Total diagnoses made Tests Dx Yield Tests Dx Yield Tests Dx Yield Test Dx Yield Tests Dx Yield s

1 86 27* 31% 59 3 5% 56 9 16% 28 0 0% - - 39

2 86 27* 31% 59 3 5% 56 9 16% 28 0 0% 47 10* 21% 49

3 86 27* 31% 59 3 5% 56 9 16% 19 0 0% 47 10* 21% 49

4 86 27* 31% 59 3 5% 45 8 18% 19 0 0% 56 11* 20% 49

5 86 20^ 23% 47 2 4% 45 8 18% 19 0 0% 66 19^ 29% 49

6 86 20^ 23% 40 2 5% 45 8 18% - - 66 19^ 29% 49

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7 86 20^ 23% - - 45 8 18% - - 66 19^ 29% 49

* In these scenarios, diagnoses suspected clinically at Tier 1 confirmed with single (or small panel) gene testing and were considered within the diagnoses made on Tier 1

^ In these scenarios, diagnoses suspected clinically at Tier 1 were confirmed with WES, as in these scenarios, WES immediately followed Tier 1 in the diagnostic pathway. These diagnoses were considered to have been made on WES

Scenario Tier 1 Tier 2 Repeat MRI Tier 3 WES Total diagnoses made

Tests Dx Yield Tests Dx Yield Tests Dx Yield Tests Dx Yield Tests Dx Yield

1 86 27* 31% 46 3 7% 39 9 23% 25 0 0% - - 39

2 86 27* 31% 46 3 7% 39 8 21% 25 0 0% 27 6* 22% 44

3 86 27* 31% 46 3 7% 39 8 21% 19 0 0% 27 6* 22% 44

4 86 27* 31% 59 3 4% 28 7 25% 19 0 0% 39 8* 21% 45

234 5 86 20^ 23% 33 2 6% 28 7 25% 19 0 0% 53 18^ 34% 47

6 86 20^ 23% 24 2 8% 33 7 21% - - 53 18^ 34% 47

7 86 20^ 23% - - 33 7 21% - - 53 18^ 34% 45

* In these scenarios, diagnoses suspected clinically at Tier 1 confirmed with single (or small panel) gene testing and were considered within the diagnoses made on Tier 1

Figure 7.1 Diagnostic pathway without whole exome sequencing

Number of infants tested (blue numbers) and number of infants diagnosed (red numbers) with modelling of the current diagnostic pathway if all 86 infants with no aetiology known prior to epilepsy onset are investigated until aetiology identified or reach end of the pathway

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Figure 7.2 Diagnostic pathway with whole exome sequencing

Number of infants tested (blue numbers) and number of infants diagnosed (red numbers) with modelling of WES performed between Tiers 1 and 2 of the current diagnostic pathway if all 86 infants with no aetiology known prior to epilepsy onset are investigated until aetiology identified or reach end of the pathway

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Figure 7.3 Diagnostic pathway without whole exome sequencing (version 2)

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Number of infants tested (blue numbers) and number of infants diagnosed (red numbers) with modelling of the current diagnostic pathway if all 86 infants with no aetiology known prior to epilepsy onset are investigated only if seizures are ongoing until aetiology identified or reach end of the pathway

Figure 7.4 Diagnostic pathway with whole exome sequencing (version 2)

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Number of infants tested (blue numbers) and number of infants diagnosed (red numbers) with modelling of WES performed between Tiers 1 and 2 of the current diagnostic pathway if all 86 infants with no aetiology known prior to epilepsy onset are investigated only if seizures are ongoing until aetiology identified or reach end of the pathway

7.3.2 Costing analysis The total cost of the modelled current standard diagnostic pathway for 86 infants whose cause was unknown at epilepsy onset was $887,386, with a cost per diagnosis of $22,754. The cost increased with the addition of WES at the end of the current diagnostic pathway to $990,786, although with 10 additional diagnoses made and a lower cost per diagnosis of $20,220. The total cost reduced as WES was brought earlier in the diagnostic pathway, albeit still higher than the cost without WES. WES at step 2 of the diagnostic pathway had a comparable cost to Scenario 1, with 10 additional diagnosis and a reduction in cost per diagnosis of more than $4000 to $18,300. Savings to the total cost appeared with removal of Tier 3 metabolic testing, without an overall loss of yield. Further saving was obtained by also removing Tier 2 testing, reducing the total cost to $609,439 and a cost per diagnosis of $12,997. Thus, if Scenario 7 were to be the new standard diagnostic pathway, over a quarter of a million dollars would be saved in diagnostic costs over three years for 86 infants. The costs of each these scenarios are listed in Table 7.9 and 7.11 below, and the additional and saved costs in Table 7.10 and 7.12.

It is important to note that two fewer diagnoses were made in Scenario 7 than in Scenarios 2- 6. As noted above in the Modelled Diagnostic Pathway section though, it is highly likely that Tier 2 investigations (which resulted in the suspected diagnosis being made) would have been performed in those two infants even if not part of the standard diagnostic pathway.

The results of investigating only infants with ongoing seizures are similar to that of investigating all infants with respect to the relative costs of each scenario. While 2-5 fewer diagnoses were made (depending on the scenario), the overall cost and the cost per diagnosis was lower than if all infants continued through the diagnostic pathway because fewer infants were studied.

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Table 7.9 Cost and yield of diagnostic pathways if all 86 infants with unknown cause at the time of presentation are investigated until aetiology identified or reach end of diagnostic pathway

Cost per Diagnoses Cost per Cost per Scenario Step 1 Step 2 Step 3 Step 4 Step 5 Total cost incremental made diagnosis patient diagnosis

1 Tier 1 Tier 2 Repeat MRI Tier 3 - $887,386 39 $22,754 - $10,318

2 Tier 1 Tier 2 Repeat MRI Tier 3 WES $990,786 49 $20,220 $10,340 $11,520

3 Tier 1 Tier 2 Repeat MRI WES Tier 3 $919,527 49 $18,766 $3,214 $10,692

4 Tier 1 Tier 2 WES Repeat MRI Tier 3 $922,252 49 $18,821 $3,487 $10,724

5 Tier 1 WES Tier 2 Repeat MRI Tier 3 $896,676 49 $18,300 $929 $10,426

240 6 Tier 1 WES Repeat MRI Tier 2 - $737,829 49 $15,058 -$14,956 $8,579

7 Tier 1 WES Repeat MRI - - $609,439 47 $12,967 -$34,743 $7,087

Table 7.10 Additional and saved costs per scenario if all 86 infants with unknown cause at the time of presentation are investigated until aetiology identified or reach end of diagnostic pathway

Scenario Costs saved through avoidance of tests Additional cost Total of multigene additional Tier 1 Tier 2 Repeat MRI Tier 3 Confirmation Total testing cost of suspected diagnoses on Tier 1 and 2

1 $0 $0 $0 $0 $0 $0 $0 $0

2 $0 $0 $0 $0 $0 $0 $103,400 $103,400

3 $0 $0 $0 -$71,260 $0 -$71,260 $103,400 $32,141

4 $0 $0 -$23,100 -$64,134 -$1,101 -$88,335 $123,200 $34,865

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5 $0 -$35,181 -$23,100 -$64,134 -$13,496 -$135,911 $145,200 $9,289

6 $0 -$58,635 -$23,100 -$199,527 -$13,496 -$294,758 $145,200 -$149,558

7 $0 -$172,973 -$23,100 -$199,527 -$27,548 -$423,147 $145,200 -$277,947

Table 7.11 Cost and yield of diagnostic pathways if all 86 infants with unknown cause at the time of presentation are investigated until aetiology identified only if seizures are ongoing

Cost per Diagnoses Cost per Cost per Scenario Step 1 Step 2 Step 3 Step 4 Step 5 Total cost incremental made diagnosis patient diagnosis

1 Tier 1 Tier 2 Repeat MRI Tier 3 - $785,070 39 $20,130 - $9,129

2 Tier 1 Tier 2 Repeat MRI Tier 3 WES $844,470 44 $19,193 $11,880 $9,819

3 Tier 1 Tier 2 Repeat MRI WES Tier 3 $801,714 44 $18,221 $3,329 $9,322

4 Tier 1 Tier 2 WES Repeat MRI Tier 3 $803,913 45 $17,865 $3,141 $9,348

5 Tier 1 WES Tier 2 Repeat MRI Tier 3 $784,205 47 $16,685 -$108 $9,119

242 6 Tier 1 WES Repeat MRI Tier 2 - $637,121 47 $13,556 -$18,494 $7,408

7 Tier 1 WES Repeat MRI - - $555,639 45 $12,348 -$38,238 $6,461

Step 1 performed at epilepsy onset, step 2 only performed if seizures ongoing >1 month, step 3 only performed if seizures ongoing >3 months, steps 4 and 5 only performed if seizures ongoing >6 months

Table 7.12 Additional and saved costs per scenario if all 86 infants with unknown cause at the time of presentation are investigated until aetiology identified only if seizures are ongoing

Scenario Costs saved through avoidance of tests Additional cost Total of multigene additional testing cost Tier 1 Tier 2 Repeat MRI Tier 3 Confirmation Total of suspected diagnoses on Tier 1 and 2

1 $0 $0 $0 $0 $0 $0 $0 $0

2 $0 $0 $0 $0 $0 $0 $59,400 $59,400

3 $0 $0 $0 -$42,756 $0 -$42,756 $59,400 $16,644

243 4 $0 $0 -$23,100 -$42,756 -$1,101 -$66,957 $85,800 $18,843

5 $0 -$38,113 -$23,100 -$42,756 -$13,496 -$117,464 $116,600 -$864

6 $0 -$67,430 -$12,600 -$171,023 -$13,496 -$264,549 $116,600 -$147,949

7 $0 -$134,861 -$12,600 -$171,023 -$27,548 -$346,031 $116,600 -$229,431

7.3.3 Commentary This costing analysis of WES in SEI simulated ‘standardised’ pathways for aetiologic diagnosis. The approach used the infants’ real diagnoses, made at the earliest possible time point in the pathway that was being modelled (rather than when the diagnosis was actually made). This approach was chosen to look at how the different diagnostic pathways would perform as a ‘protocol’ at the group level in ideal circumstances. It was chosen over individually costing the tests each infant had undergone, partly as that approach would have been prohibitively time-consuming and also made it more difficult to simulate the impact of WES at different points in a tiered diagnostic pathway. A further issue for SEI (that is not relevant in some other conditions) is that WES will not simply replace or precede standard diagnostic testing as some tests such as brain imaging provide potentially management-changing information that WES cannot (this is discussed further below). Thus, a tiered approach to diagnostic investigations is expected to be the ongoing standard; this work informs the structure of a more cost- effective and time-efficient model for reaching a diagnosis.

The analyses were performed twice –on all infants and confined to infants who are still having seizures. The clinical reality is somewhere in between these two scenarios; while some infants whose seizures cease do not have further investigations, some do - usually the infants with more severe disorders with coexistent severe developmental delay, in whom there is greater imperative to make a diagnosis for the reasons presented earlier in this chapter, or where the family is considering further children. Both analyses essentially showed the same pattern of results. So, the issue of determining how accurately the number of infants investigated in these models is estimated, compared with what would happen in clinical practice, is not critical to evaluating the performance of each pathway relative to the current standard.

It is clear that WES adds to the diagnostic yield in SEI, regardless of where it is performed in the diagnostic pathway. If WES is performed earlier in the pathway, more infants had a diagnosis made earlier in their clinical course. This included infants who would not have had a diagnosis made without WES and infants whose diagnoses were made on WES rather than later tier testing. With WES as the second step in the diagnostic pathway, the use of this technique to confirm the eight diagnoses suspected at Tier 1 that would otherwise have been confirmed by targeted gene testing was

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simulated. Using WES for this purpose was only approximately $2000 more expensive overall than the targeted testing in these eight patients, but has the advantage of being able to test other genes if the clinical suspicion was not correct or other genes for that disorder are subsequently discovered. WES is actually cheaper in some individual patients, particularly in one who had a four-gene panel performed for suspected Aicardi- Goutières syndrome. A potential issue of replacing targeted testing with WES is the possibility of ‘incidental’ or ‘secondary’ findings, but this could be obviated by initially analyzing only the gene(s) of interest. WES would not be appropriate in all in instances as some types of genetic variants, for example triplet repeat expansions, are typically not identified with this technique. Clinical judgement would dictate where alternate approaches to WES would be required.

In addition to added yield, performing WES (except when performed after all tiers in the standard diagnostic pathway) reduces the number of infants undergoing the later tiers of testing. Given the low yield of those tiers, their expense both in laboratory costs and clinician time (Tier 2 requires a clinician to perform a lumbar puncture and Tier 3 a surgeon to perform biopsies), and their invasive nature and potential for complications for the infant, this is an important advantage of WES. The earlier that WES is performed in the diagnostic pathway, the greater the number of later tier tests avoided.

There was no yield of Tier 3 testing. Two infants would have had a mitochondrial diagnosis (suspected at Tier 2) confirmed on Tier 3 testing, but in those cases, Tier 2 would have been performed separately to the prescribed diagnostic pathway (i.e. repeat MRI that is between Tiers 2 and 3 would not have been performed). Additionally, the yield of Tier 2 testing was very low. From a yield point of view, there is a clear rationale to remove Tier 3 testing from the standard diagnostic pathway, and a strong argument to remove Tier 2 given the yield is also very low. However, one treatable diagnosis was suspected on Tier 2. Given this, potential options for future diagnostic pathways would include keeping Tier 2 testing in the pathway, or removing it from the pathway undertaken by all infants, but with criteria developed to identify a subset of infants in whom it should be performed in addition to a standard pathway. The treatable aetiologies that could be diagnosed or suspected on Tier 2 testing include Glut1 deficiency and PNPO deficiency. The urgency to diagnose the latter is partly offset by the ease of implementation and tolerability of the specific treatment, pyridoxal-5-

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phosphate (Mills et al., 2014). Pre-emptive treatment with this (and other vitamins) is standard practice at our centre. In contrast, the treatment for Glut1 deficiency, the ketogenic diet is more difficult to implement. Biochemical testing for Glut1 can make a presumptive diagnosis on the day of testing, whereas genetic testing will take some weeks; delaying treatment by some weeks may adversely impact outcomes (Alter et al., 2015). The cost of Tier 2 testing is another factor that may further guide its inclusion or otherwise in the standard diagnostic pathway. This is discussed below.

WES was assessed at different points in the diagnostic pathway, but not before Tier 1 testing. There are a number of reasons for this, and discussing this issue brings up more broadly which investigations WES could not replace as they provide information that WES cannot. The most obvious is MRI, which has high and relatively immediate yield and which may be management-changing. An example of this is that of the often surgically-treatable FCD. For these infants, developmental outcomes are expected to be better if surgery can be performed, and seizures terminated, as early as possible. The surgical work-up often takes some time, as other investigations independent of diagnosis, such as more detailed MRI, other imaging such as FDG-PET, other investigations like video-EEG monitoring and baseline assessments of development and visual fields, are often needed. Thus, the earlier this can be put in place, the earlier surgery could be performed.

CMA is another Tier 1 test, which has low (albeit some) yield, and the majority of infants with a chromosomal disorder had their diagnosis made prior to epilepsy onset. While the yield is low, these disorders will not be detected by current NGS techniques, which are limited at identifying copy number variants (Retterer et al., 2015). It is possible that this may change in the future with advances in NGS techniques, and the need for chromosomal microarray obviated. In the meantime, CMA will be useful for some infants. In this study, infants with causative copy number variants either had neonatal onset of seizures, or other abnormalities present (developmental delay, hypotonia) prior to seizure onset. While it is tempting to suggest limiting this test to these infants, as well as infants with dysmorphic features or other organ abnormalities, the numbers in this study are too low to support such an approach. A further potential use of chromosomal microarray, particularly in consanguineous families, is its ability to

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detect long continuous regions of homozygosity which can highlight regions of the chromosome that may harbour pathogenic recessive mutations.

The last component of Tier 1 testing is the metabolic testing performed on urine and blood. Again this is low yield, but may detect conditions in which specific treatments may improve outcomes such as pyridoxine-dependent epilepsy (caused by ALD7HA1 mutations) and urea cycle defects quickly and at relatively low cost (McBride et al., 2004; van Karnebeek & Jaggumantri, 2015). Whether chromosomal microarray and Tier 1 metabolic testing would be cost-effective if performed separately to MRI is something that has not been looked at here, but would be worthwhile. Like Tiers 2 and 3 testing, it may be that Tier 1 metabolic and chromosomal testing could be confined to a subset of patients. In this study, all infants with a metabolic or chromosomal diagnosis were either neonates, or had developmental delay prior to epilepsy onset. These factors, as well as others such as a consanguineous family, dysmorphic features or other organ abnormalities and MRI findings suggestive of a metabolic disorder could be indications to perform these tests, but potentially only after the initial MRI has not identified an aetiology.

It is vital to consider the cost, and not simply the yield, of including WES at different points in the pathway, with and without omitting specific metabolic investigations. A population-based study can provide group-level perspective that a non-population-based study cannot; this is vital for understanding the optimal pathway of diagnostic investigation at a health systems level. Including WES at any point in the diagnostic pathway reduces the cost per diagnosis given the increased number of diagnoses made. It is not until Tier 3 or both Tiers 2 and 3 testing is removed from the pathway that approaches involving WES become cheaper overall, with no or only a small loss of yield.

Thus, it is clearly cost-effective to perform WES in infants with SEI early in the diagnostic pathway, and to remove some metabolic testing. My recommendation for the optimal future diagnostic pathway is to perform Tier 1, then WES, then repeat MRI (Scenario 7), and to drop Tiers 2 and 3. The potential loss of yield with dropping Tier 2 at a group level could be accounted for by having a list of criteria to prompt such testing in a small subset of infants. Given this group-level scenario was over $250,000 cheaper

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than the current standard diagnostic pathway, the addition of Tier 2 in a small subset of patients will still be cheaper overall.

The use of WES in clinical practice has been limited by lack of funding for this test. In Australia, WES is not currently funded through the Medicare Benefits Scheme subsidized by the Federal Government. Importantly, many Tier 2 and 3 tests, including transferrin isoforms, lysosomal enzymes, very long chain fatty acids, CSF neurotransmitters and respiratory chain enzyme analyses, are also not subsidized. Thus, the costs of both WES and much of the metabolic testing would fall to the hospital (or the patient). Given that the hospital currently funds metabolic testing, it would be in the hospital’s interest to fund WES in place of this for infants with SEI.

It is very important to recognize that there are financial costs associated with further investigating VOUS, and that these may be substantial. These were not included in this costing analysis, largely because they are very difficult to quantify, likely to be highly variable, and often incurred at a research level rather than paid for by hospitals or governments as part of clinical care. Other costs such as parental anxiety and reductions in quality of life that may be associated with this, are clearly important here too and not addressed in detail by this study.

There are a number of future directions to pursue to further optimize diagnostic pathways in SEI. Tier 1 testing may be able to be further rationalized, with some or all of the chromosomal and metabolic testing performed only in a subset of patients. It may be that the optimal diagnostic pathway may be different for different epileptic syndromes. For example, infants with spasms and unifocal seizures are more likely to have a malformative disorder than infants with neonatal/early infantile epileptic syndromes. For the former, a pathway with a greater emphasis on imaging and less or no emphasis on metabolic testing may be appropriate. Finally, the role of WES or other genetic testing to confirm the genetic basis of metabolic and structural conditions needs to be considered. It is unlikely that the same WES gene panel is the most appropriate genetic test for these infants, and that other approaches employing next-generation techniques that allow deeper sequencing and enable detection of the somatic mutations (for example in malformative disorders in which mosaic mutations are increasingly recognized) are likely to be preferential (Jamuar et al., 2014).

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Chapter 8: Discussion

8.1 Introduction

While there have been other population-based studies of specific infant epileptic syndromes and all infant epilepsies, this is the first population-based study of the ‘severe epilepsies of infancy’. Among infants with epilepsy, this is the patient group that it is critical to better understand – the cause remains unknown in many, effective treatment is often not available, the clinical outcomes are frequently devastating and the health burden massive.

It is likely that outcomes will improve with better seizure control and targeted treatment of the underlying aetiology. Development of novel and targeted therapies will be developed based on knowledge of the causes of SEI. Maximising outcomes requires early implementation of effective treatment, for which early aetiologic diagnosis is vital. This study is timely given the recent explosion in genetic causes of this group of disorders has enabled an aetiological basis to be established in many more infants.

This study provided thorough and detailed analysis of the epidemiology, aetiologies, epileptology and outcomes of these conditions, and investigated the cost and utility of diagnostic investigations including WES. It has:

 shown that SEI is relatively common for a group of severe disorders  established the incidence of some epileptic syndromes for the first time  further delineated the electroclinical features in infants whose epilepsy does not fit into one of the well-described epileptic syndromes  identified FCD as the most common aetiology and channelopathies the most common group of ‘genetic’ disorders  determined that high quality imaging and genetic testing increases the proportion of infants in whom the aetiology is identified from just over half to two-thirds of cases and  shown that implementation of WES and removal of some metabolic testing in diagnostic pathways improves diagnostic yield for less cost, informing strategies to improve use of health services.

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The following sections discuss the design of the study in achieving these goals, and present the major findings and their implications, together with the study’s limitations.

8.2 Study design

The population-based nature and completeness of ascertainment in this study is a major strength of its design, and provides new insights that non-epidemiologic studies do not; namely the ability to understand the relative importance of each aetiology, epileptic syndrome and diagnostic test across the whole SEI population. It enables the beginnings of an understanding of prognostic factors, outcomes and burden of disease with currently available therapies, and provides a ‘historic’ comparator to compare outcomes as new treatments emerge. The completeness of ascertainment was aided by both the severity of these conditions and the centralised medical care in Victoria; that is, infants will virtually always present to medical care and, when they do, there are a limited number of centres at which they are managed. This type of study would not have been feasible in many other parts of the world, nor in less severe conditions, as presentation to medical attention may not be universal, nor occur in a uniform fashion to a specific specialty group such as paediatric neurologists, as is the case for the severe epilepsies in infants in Victoria.

The depth in which each infant’s data was studied by a single (or small number of) investigators, as well as the close-to-complete recruitment of infants with unknown aetiology for genetic testing , affords this study consistency and accuracy that is lacking in studies that do not take this approach. Two important examples are that firstly, brain imaging review identified FCDs not reported clinically, and secondly, review of interictal EEGs in all infants, and seizure videos in most, allowed a greater degree of accuracy in classifying seizure types and epileptic syndromes, as this can be very difficult in infants on the basis of seizure descriptions alone.

There are several limitations of this study, all of which would require longer-term study of this cohort, or study of a new incident cohort, to overcome. Standardised developmental assessments were not performed. Developmental impairment was often so significant as to easily allow distinction between those with severe delays and those whose deficits were milder. But, comparison of the developmental level (relative to age- expected) within each individual over time is limited, and very mild deficits may have

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been missed. The latter may be partly overcome by longer-term study of outcomes, as subtle deficits will become more apparent with age. Additionally, longer-term study will allow determination of the rate of autism spectrum disorders in this group as it was not possible to do that here. Accurate study of developmental trajectory over time would require the study of a new incident group prospectively. This study is being extended (over additional years) to collect a larger population-based group; this could be implemented in newly-identified infants. Finally, while extensive study of aetiologies was performed, there are some infants who have a suspected diagnosis that could be made on currently available clinical or research testing, but such testing was not performed. This includes a number of infants with suspected FCDs who have not had repeat brain imaging (mainly as seizures have stopped), as well as an infant with Dravet syndrome who has not had SCN1A sequencing performed as yet. This may be remedied in the future, particularly in the case of those with suspected FCD who may have recurrent seizures that prompt additional investigation.

8.3 SEI definition

A potential criticism of this study is that a non-standard definition was used for this patient group. Although clinicians can easily identify an infant with ‘severe epilepsy’, the term ‘SEI’ and the specific definition use here is not widely used. While this is not ideal for comparison across different studies (particularly those with an epidemiologic basis), there were several reasons why this definition and term were chosen, as discussed extensively in the Literature review and Methods chapters.

It is important then, to consider whether the term did, in fact, cover the whole group of infants whose epilepsy was ‘severe’. An important gauge of that is whether any infants with the well-defined epileptic syndromes that are considered severe were excluded. As predicted, some infants with Dravet syndrome did not meet inclusion criteria, but all infants with the other infant-onset ‘severe’ epileptic syndromes were included. Given the exclusion of some with Dravet syndrome, this definition is better thought of as encompassing the group of infants with frequent seizures during infancy rather than those with infant-onset epilepsies that will be severe at some point, but not necessarily before 18 months old. Were this definition to become more widely used, it may be that some would prefer that those with Dravet syndrome were an automatic inclusion in the same vein as those with infantile spasms, although the inclusion of this group with 251

infant-onset later-severe epilepsies, but not others that could potentially behave in a similar same manner, such as structural focal epilepsies, may seem arbitrary. Comparison of the performance and utility of alternative definitions of SEI, such as one that has Dravet syndrome as an automatic inclusion, were not performed in this study, but are of interest.

Outside of the ‘severe’ epileptic syndromes, a group of infants, not covered by this definition, whose seizures may be considered severe are those with recurrent status epilepticus (with no or infrequent seizures between bouts of status) such as some infants with Dravet syndrome, PCDH19 encephalopathy and Sturge-Weber syndrome (Nabbout & Juhasz, 2013; Trivisano et al., 2016). Again, this is a patient group that some would consider should be included in a definition of severe epilepsy. It was not included here for a number of reasons, including concern about compromising the completeness of ascertainment of infants meeting a definition of SEI that included this criterion as not all infants with febrile status epilepticus (even when recurrent) are referred for an EEG. Additionally, if episodes of status epilepticus are solely febrile then that infant does not technically have epilepsy.

Another measure of the performance of the study definition is whether it did in fact denote a group with poor outcomes. Overall, outcomes for seizure freedom, development and survival were indeed very poor. Normal development at two years old was seen in less than 10% of the group. Within those with normal development were some infants with IS of unknown cause with prompt response to treatment (and no relapse). This group of patients has been previously recognised in a number of studies to have a more favourable outcome (Kivity et al., 2004); thus, their inclusion in a ‘severe’ cohort should be questioned. In establishing the study definition, having a definition that can be applied early in the epilepsy was felt to be critical. Infantile spasms were deemed an automatic inclusion criteria as the overall rate of poor outcome is very high, and it is often not apparent early which infants will have a normal outcome. Given that this inclusion means a small proportion of the SEI group is expected to have a normal outcome, the definition should be considered to denote infants who have a ‘potentially’ severe epilepsy of infancy, with very high but not universal rate of poor outcome.

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An important departure from the most widely used term for ‘severe’ epilepsies, EE, was that the definition of SEI did not include a developmental criterion. Although that criterion conveys a very important inference, it is often difficult to disentangle the developmental effects of the epilepsy from those of the underlying condition. In choosing to focus solely on epilepsy factors, it was hoped that this definition would overcome that issue, but still predict poor outcomes among infants with seizures. Certainly, infants meeting the definition of SEI had a very high rate of poor epilepsy and developmental outcomes and a high risk of death, showing that it does in fact select a group with poor outcome.

In considering the usefulness of this definition though, two very important questions are whether infants with epilepsy have aspects of their epilepsy that are easily split into two groups (i.e. ‘mild’ vs ‘severe’ epilepsy) rather than lying on a spectrum of severity without an obvious point of division, and whether infants with epilepsy who do not meet criteria for SEI actually have better outcomes. These were not directly addressed in this study as infants without SEI were not studied in detail. It is clear though that there is a point of distinction in seizure frequency between infants with SEI and those without, which is in favour of there broadly being two separate groups. All infants with SEI easily met the seizure frequency inclusion criterion, most clearly exceeding it with multiple daily seizures. In contrast, few infants without SEI (data not shown) came close – a few had daily seizures for a few days (but not a week), and some had almost weekly seizures for a period of time, but most had significantly lower seizure frequency than the cut-off in the definition. This data is similar to that of a study by Datta and Wirrell of infants age 1-12 months with epilepsy that clearly showed a split into two groups with infrequent (< monthly) and frequent (> weekly) seizures, rather than a broader spectrum of frequency (Datta & Wirrell, 2000). Determining whether these two groups clearly segregate good and bad outcomes will require further study. One would speculate that, overall those with infrequent seizures will do much better. However, while some will have resolution of epilepsy and normal developmental outcome (e.g. those with ‘benign’ neonatal and infantile epileptic syndromes), it is expected that this group will also include some with problematic epilepsy later in life (e.g. Dravet syndrome, some with brain malformations) and abnormal development (e.g. some chromosomal abnormalities associated with infrequent seizures and intellectual disability); use of non-epilepsy factors in prognostication may also be required.

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8.4 Epidemiology

With an incidence of 51.2/100,000 live births/year, or approximately 1:2000, SEI is relatively common for such a severe group of disorders. Furthermore, this figure belies the importance of SEI with respect to health burden. This is evidenced by large studies of paediatric hospital inpatient costs in the US, which suggest that a significant proportion of hospital costs are incurred by infants with seizures and neurologic impairments (Berry et al., 2012; Standridge & Horn, 2012). Family and societal costs of these conditions are also immense, but difficult to measure. Thus, improvements in diagnosis, treatments and outcomes are likely to have a bigger impact at a societal level than the incidence of SEI would suggest.

In addition to studying the epidemiology of the whole group of SEI, this study was also interested in the incidence of particular groups of SEI. It is the first study to determine the incidence of the epileptic syndromes EME and EIEE, and one of the first to study the incidence of EIMFS. It has confirmed the former to be extremely rare and shown the latter two to be more common than perhaps anticipated, with incidences not much lower than the reported incidence of Dravet syndrome. The incidence of EIMFS in this study is considerably higher than previous estimates, perhaps due to the large number of ictal recordings available to confirm the diagnosis in these infants.

Until now, there has been no study of the genetic epidemiology of SEI, which is needed to understand the relative importance of particular single gene disorders. Given the genetic heterogeneity of SEI, a large group would be required to determine the relative frequency of individual causes, given that even commonly identified genes have been estimated to account for only 1-2% of patients (Carvill, Heavin, et al., 2013). While this patient group is not of a size at which that can be achieved, it does begin to get at which groups of genetic disorders are most important in this age group, with channelopathies seen in over half of infants in whom a genetic cause was identified, almost solely in infants with EIEE and EIMFS (and variants), and Dravet syndrome. Such findings are highly useful for identifying research priorities, but are also useful at the clinical level with respect to identifying likely genetic diagnoses, and potentially beneficial treatments. For example, improvement with sodium channel blockers has been reported for three genes that can cause EIEE and/or EIMFS, KCNQ2, SCN2A and SCN8A. Thus, for those two syndromes, consideration should be given to an early trial of sodium 254

channel blockers, potentially even before a genetic diagnosis is made. It is important to mention that no CDKL5 or STXBP1 mutations were identified in this group, which is perhaps surprising given they are considered important infant epilepsy genes, with one study showing STXBP1 mutations in 30% of a group of infants with EIEE (Saitsu et al., 2008) and 10% of early onset epileptic encephalopathies that do not fit an established epileptic syndrome (Deprez et al., 2010). There are two possible explanations for this. First, that some infants in this group do have mutations in these genes that were not detected by the techniques used. Secondly, that issues such as referral and reporting bias and chance may account for this – for example, small non-population based studies may overestimate the proportion of infants with a particular cause, and a larger population- based study than this one may be required to detect an infant with that cause. It is worthwhile noting that study of Victorian infants with SEI born in 2014- (an extension of this study that is underway, with approximately 200 individuals with SEI born 2011- 2015 now identified) had identified one infant with a CDKL5 mutation and one with a mutation in STXBP1.

8.5 Aetiologies

The aetiology of SEI was identified in two-thirds of infants. Fifteen infants, or 28% (15/53) of the group with no diagnosis after clinical testing, had an aetiology identified on research testing. A further nine infants have a suspected diagnosis, and a number of infants have variants of unknown significance identified on WES that require further study to determine whether they are pathogenic. Thus, significant inroads are being made into determining previously unknown aetiologies, although there remains some way to go.

As expected, many different aetiologies were identified. The make-up of aetiologies had some similarities with, but also differences from, previous cohorts. Some aetiologies reported to be common in previous studies of infant epilepsies, such as TS and trisomy 21, were also among the most common causes in this study. Some groups of conditions reported at relatively high frequency in previous studies, were seen less commonly here. The most important example is that of acquired aetiologies, seen in just 12% of infants in this study. Here, the majority of infants had a genetic or presumed genetic basis for SEI. And, some conditions not detected at high frequency in prior studies such as genetic disorders and subtle brain malformations were seen commonly in this group, 255

largely reflecting advances in diagnostic imaging and genetic understanding influenced by new technologies. With an ability to now identify these groups of conditions, this study gets closer to establishing the true spectrum of aetiologies of SEI.

Brain malformations were the most common aetiologic group, diagnosed in 26% of infants, with unifocal malformations such as FCDs and multifocal malformations such as TS predominating. Malformations, particularly FCDs, are under-recognised. Identification of subtle malformations in this study, that had not been detected clinically, underscores the importance of detailed review of brain imaging guided by clinical information such as seizure semiology, EEG focality and focal findings on neurologic examination. Despite these additional diagnoses, FCDs almost certainly remain under-diagnosed in this cohort, suggesting a need for improved imaging technologies or repeated brain imaging in optimising diagnosis in this group. Focal brain malformations are among the most important diagnoses to make given that epilepsy surgery can improve outcomes, with seizure freedom and improved development possible in some cases. While there is much focus on the (undoubtable and very important) need to develop new treatments for SEI, improving utilisation of currently available treatment is a more neglected area. Early use of epilepsy surgery is likely to be the highest impact intervention in infants with SEI; optimising its use in infants with brain malformations is a priority. This will require adequate brain imaging (and re-imaging), expert review of scans, and early referral to epilepsy surgery for determination of suitability for surgery.

Single gene disorders (non-malformative, non-metabolic) disorders were identified in 15% of all infants with SEI, and just under 30% of those in whom standard clinical investigations, brain imaging, metabolic and chromosomal testing, would not have made an aetiologic diagnosis, tallying with estimates of the yield of WES in non- population-based studies. At this point though, the genetic bases of SEI have been incompletely investigated, and the number with a ‘genetic’ cause will be higher than identified here. Firstly, only genes reported in infants with seizures have been tested; mutations in new genes remain likely; unbiased analysis of WES data is planned to investigate this possibility. Secondly, particular types of mutations are not detected (either well or at all) by WES, including indels, triplet repeat expansions and intronic variants; methodologic improvements or different NGS techniques will be required to

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address this. Thirdly, the role of mosaic mutations, which are increasingly recognised in the literature, has not been addressed well here. Very low level mosaicism will not be detected by the WES technique used in this study, and mosaic mutations isolated to brain tissue not detected at all. The role of mosaic mutations in brain tissue in epileptogenic brain malformations is now well-established. It is possible, perhaps even likely, that mosaic mutations play a role in non-malformative epilepsies, although lack of access to brain tissue in these infants limits investigation of this possibility.

Finally, it is clear from this study that the group with unknown cause for SEI contains not only infants with ‘genetic’ disorders, but also infants with occult FCDs which likely also have a genetic basis, which may be due to germline or mosaic mutation(s).

8.6 Clinical features

This study confirms findings of previous studies with respect to a number of aspects of the epilepsy. Spasms and focal seizures predominate, with generalised seizures (especially absence, atonic and generalised tonic-clonic seizures) seen infrequently. West syndrome is the most common epileptic syndrome; it and variants being seen in approximately two-thirds of infants at onset or evolution of the epilepsy.

There are some important aspects of the epilepsy though for which this study builds on previous knowledge. Here, we were able to classify the epilepsies in those with ‘complex’ phenotypes that do not fit a well-described syndrome by determining the syndrome is most closely resembles (e.g. EIMFS-like, Dravet syndrome -like). It is expected that there will be differences in treatment-responses, outcomes and aetiologies across different phenotypes within this ‘complex’ group (for example, epilepsies associated with myoclonic and tonic seizures and generalised spike-wave on an EEG are likely to behave differently from those with focal seizures) and that this will be clinically useful. Further validation of such an approach is required to determine if this is indeed the case, and also if these ‘variant’ syndromes behave similarly to the ‘prototypic’ syndromes.

A second important advance stems from increasing the number of infants with an aetiologic diagnosis, allowing a more complete dissection of the aetiologies associated with each epileptic syndrome. The most notable of these has been discussed previously,

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namely that structural aetiologies are even more important than previously shown in infants with spasms and unifocal epilepsies, and are underdiagnosed.

Outcomes in those with neonatal and early infantile epileptic syndromes is very poor; despite often aggressive treatment, all infants had severe developmental delay or had died by two years old. In contrast, normal outcomes were seen in a proportion of infants with West syndrome and variants, and some infants with unifocal epilepsies. These differences in outcomes between subgroups of infants highlight that prognostication is better considered at a subgroup level of epileptic syndrome or aetiology given outcomes are not uniform across the whole group. Similarly, if this group is used as ‘historic’ controls for future outcome-based research, in particularly in considering whether outcomes with novel treatments are better than what was historically seen, the differences in outcomes between subgroups of infants should be taken into account.

8.7 Diagnostic investigation

This study clearly demonstrated an increased diagnostic yield with high quality brain imaging and WES.

From this and previous studies, there is no doubt that WES (or other NGS technologies) has a significant role in diagnostic investigation in SEI. Until now though, its cost effectiveness in SEI had not been demonstrated. This is the first population-based health economic study of WES in SEI. It shows that performing WES early in the diagnostic pathway, and reducing the use of metabolic testing, results in more diagnoses for less cost. Thus, there is a clear financial as well as clinical argument for the introduction of WES into routine testing. This study does not consider indirect cost savings in areas unrelated to diagnostic testing that arise as a result of making additional diagnoses. Where a diagnosis results in management changes that improve the infant’s condition, there are likely to be savings in hospital inpatient costs, and economic benefits such as those that result from a parent being able to return to work. Other financial savings may apply even where there is no direct benefit to the infant, such as the costs saved by avoiding a second affected child in the family. Such savings are difficult to measure, but potentially considerably larger. Thus, considering diagnostic costs alone considerably underestimates the financial and social benefits of improved diagnostic yield.

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It is important to note that the economic analysis in this study did not consider the cost of further investigating variants of unknown significance identified on WES. Given that such variants are being further investigated in some study patients, the total cost of this is not yet known. This warrants further investigation however, as there is no doubt that these variants are associated with additional financial costs, as well as the psychosocial costs that uncertainty can bring to bear on families.

There is also a clear argument for limiting all but the very basic metabolic testing at the group level given the high cost, invasiveness and low yield of these tests. Metabolic disorders are not common and, in many cases, WES could be expected to make the diagnosis and obviate the need for many biochemical investigations. There are a number of instances in which metabolic testing still has a place. In cases where there is a time- critical nature to making a diagnosis, namely if a treatable metabolic diagnosis is possible or where the severity of the condition is such that palliative approaches are being considered (e.g. in neonates). In these cases, if the turn-around-time for the diagnostic biochemical test is considerably less than for the genetic test, it should be performed. Clear guidelines for targeted use of metabolic testing in SEI should be included in diagnostic protocols.

With time, further technical advances and greater knowledge of the aetiologies in infants whose cause is currently unknown, the optimal group-level diagnostic pathways will change. More advanced NGS technologies such as WGS will more reliably detect copy number variants, and chromosomal microarray testing may become redundant. Currently, WGS is rarely used in the clinical realm as limitations in knowledge of the normal genetic variation outside the exome mean that determining pathogenicity of non- exonic variants is difficult and the analysis remains challenging (Precone et al., 2015). However, as this knowledge improves, WGS is likely to replace WES given it can detect variants both in and outside of the exome.

Two aspects of an optimal diagnostic pathway that were not investigated here bear further thought. The first is the optimal means of confirming the genetic basis of malformative and metabolic diagnoses. Targeted analysis of exome data may be a pragmatic approach – the cost of WES is not substantially higher than that of single gene (at least for larger genes) or small gene panel testing. Initial analysis could be limited to the gene or genes of interest, thereby avoiding the possibility of incidental 259

findings. Where this does not identify a cause, the exome could be analysed more broadly without incurring additional sequencing costs.

The second is that a ‘one-size-fits-all’ approach to diagnosis may not be the best option. The data from this study suggests that a high proportion of infants with spasms and unifocal epilepsies have a structural basis for their SEI. It may be in these infants that an ideal diagnostic pathway would focus more on imaging and less on metabolic testing. Further work will be required to determine whether this is indeed the case.

8.8 Significance of study findings

While this study investigated many aspects of SEI including incidence, clinical features and outcomes, much of the study was aimed at understanding the aetiologies of SEI and how to maximise early diagnosis. It is in the knowledge acquired in these parts of the study that benefits are most readily demonstrated, and can be immediately translated into clinical practice.

Improved diagnostic yield through use of high quality brain imaging and WES has multiple potential benefits at the individual patient level. In some instances, a diagnosis impacts on treatment, whether a specific targeted and potentially curative therapy such as epilepsy surgery for FCDs, or rationalisation of antiepileptic drugs. The earlier that diagnosis can be made, the earlier optimal treatment can be implemented, with greater benefit. Understanding the aetiology of SEI also affords a better understanding of prognosis and allows screening for complications, both of which have implications for quality of life.

At the family level, making a diagnosis has a number of major implications. It brings the so called ‘diagnostic odyssey’ to an end, providing an explanation for the infant’s condition, sometimes alleviating feelings of blame or guilt that parents may carry. It allows parents to benefit from disease-specific support groups and early access to new information. Critically, it allows accurate reproductive counselling where this is desired, to prevent a second affected child in the family. This is highly relevant in SEI, as many parents are young and would like more children.

At a broader level, determining the role of particular diagnostic investigations in SEI has informed improvements in diagnostic pathways which can now be implemented

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into clinical practice with direct cost savings to the health system. A better understanding of the aetiologies of SEI, including which are more important at the group level, will guide future research efforts into developing novel treatments.

8.9 Unanswered questions and future directions

There remain many aspects of SEI that are not well understood; these knowledge deficiencies need to be overcome to improve outcomes in these devastating conditions.

The aetiology remains unknown in one-third of infants, and diagnostic investigation could be further optimised. Further work is required in these areas and will be a focus of research following on from this study. Genetic testing techniques such as unbiased analysis of WES data and WGS, and imaging technologies such as 3T MRI-PET will be utilised to identify aetiologies in infants with still unknown cause, and the yield of these tests in SEI determined.

The genetic basis of brain malformations will be studied in more detail, particularly to determine the proportion of infants with FCDs who have an underlying germline mutation. Germline mutations have been recently demonstrated, including some with variable penetrance and severity that can be seen in multiple members of a family, but it is not clear what proportion of those with FCDs that this is relevant to. Given the reproductive counselling implications, this is an important area to better understand.

The ‘genetic’ aetiologies will be further investigated given these are a major cause of SEI and almost universally require better treatments than are currently available. Areas including genetic epidemiology and genotype-phenotype correlations have been studied to a degree here, but require further research and larger patient groups to better determine the most important genetic aetiologies in SEI and their associated clinical features. This information will be of significant use to clinicians, and to laboratory colleagues who are working on developing targeted therapies for particular causes of SEI. This is the most likely path to transform the outcomes of these devastating disorders in the future.

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Appendices

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Appendix A: Single gene causes of infant epilepsy1

WS  LGS most females had cerebral deceased, no proteins AD (de novo) Non-specific Refractory SSW, MF Moderate- Optic atrophy normal atrophy other Sz - ES, F, T, severe DD transferrin reported 7 (one male) aT, M females isoforms (one deaths reported male Timal 2012 had abnormal DeLigt 2012 transferrin Epi4K 2013 isoforms and Michaud 2014 multiorgan Hino-Fukuyo abnormalities) 2015 Smith-Packard

2015 Dimassi 2016 ARX expansions 0-7 m EIEE IS, T Hyps, B-S - DD in most Movement Micropenis in Normal in 50% Some Transcription factor WS disorder (most some reported crucial for interneuron X-linked (males Non-specific Ongoing Sz N, MF Severe- commonly Non-specific deaths migration and affected) in some - T, profound DD dystonia +/- abnormalities in function M or F, episodes status 50% incl. basal >30 some Sz dystonicus) ganglia

1 Table adapted from supplementary table in McTague and Howell et al, Lancet Neurology 2016. Original table made by me and Amy McTague. This revised version of the table includes new genes and additional information on genes included in the previous table.

Gene Age Sz Epilepsy Sz at onset EEG at Notable Development Other features – Other features Magnetic Survival Protein function and onset syndromes onset treatments at onset neurologic – non- resonance mechanistic Inheritance (% of cases of Sz neurologic brain imaging implications this evolution EEG Development Cases with severe syndrome evolution evolution epilepsy reported where known) References free after cavitation/ Stromme 2002 infancy signal change, Guerrini 2007 cerebral Kato 2007 atrophy BRAT1 0-9m EIEE T, M, C, F B-S, MF B: ZNS (2 N/A in most, DD Neonatal rigidity Dysmorphism Normal or mild Death in DNA repair and (most EOEE patients) in some Hypertonia frontal lobe infancy in > apoptosis AR in Non-specific Refractory N/A Dysautonomia hypoplasia or half of week Sz Profound DD Microcephaly cerebral reported 18 1) atrophy cases, survival into Puffenberger 2012 childhood in Saunders 2012 remaining Saitsu 2014 cases Straussberg 2015 322 Srivastava 2016 Smith 2016 CACNA1A 0m-4y EOEE M, T, TCS, MF B: LTG, VPA in DD in most Episodic or - Normal or Some Voltage-gated ion EIMFS IS, A, aT, F Hyps patients with persistent cerebellar reported channel AD de novo or (some GGE with GSW (>3Hz GGE Mod-profound nystagmus and atrophy or deaths predominantly inherited with 0-1m, refractory Refractory and <3Hz), phenotype DD ataxia (sometimes diffuse atrophy expressed in neuronal variable severity most absences +/- Sz (multiple MF, PFA Autistic improved with tissue in family, AR (1 sib others other seizures types), features in ACZ) pair only) 4-18m) WS (reported episodes of some in status Hypotonia or 14 microdeletion epilepticus hypertonia s involving (CSE or Auvin 2009 CACNA1A) NCSE) in Epi4K 2013 LGS most, reflex Family members Hino-Fukuyo 2015 seizures in may be more Damaj 2015 some mildly affected Byers 2016 with episodic Reinson 2016 ataxia +/- epilepsy Epi4K 2016 Also causes non-EE epilepsy: FS, absences, other GGE

CHD2 9 m -4 Non-specific M, A, GTCS, GSW, PSW, - DD in most, Ataxia - Normal or - Chromatin y A(EM), ‘aT- activates in vermis remoderateeler, AD (de novo) M-A’, sleep Mild-severe DD hypoplasia specific function prominent Autistic unknown >30 clinical SSW, MF features in photosensit some Carvill 2013 ivity Suls 2013 Chenier 2014 Refractory Thomas 2015 Sz - as above plus T, aT, NCSE DNM1 2-13 m WS IS Hyps, MFD - DD in some Hypotonia - Normal or - GTPase involved in WS LGS cerebral activity-dependent AD (de novo) Refractory SSW, PFA, Severe- atrophy synaptic vesicle Sz in most MFD profound DD formation at inhibitory 9 - T, aT, F, synapses. Mutations atA, M predicted to impair Epi4K vesicle recycling and Consortium 2013 reduce T firing at EuroEPINOMICS synapses 2014

324 Refractory Aggressive translation. Also

De Ligt 2012 Sz in many behaviour activates Akt, and may Veeramah 2013 have a roles in Nakajima 2015 multiple other cellular Inui 2016 functions Lopes 2016 Lam 2016

FHF1 2d- Non-specific F, T, M, TCS MF B: PHT? UK Hypotonia Severe Cerebellar Two reported Fibroblast growth 6wks Ataxia (episodic constipation atrophy deaths factor which interacts AD Refractory MF, hyps Mod-profound or persistent) Feeding with and modulates Sz (without DD difficulties voltage-gated sodium 5 spasms) channels

Siekerska 2016 Al-Mehmadi 2016 FOXG1 mutations 3 m-3 y Non-specific F, M, T, TCS F, MF - DD Rett-like features Mild postnatal Gyral - Transcriptional in most (‘congenital growth simplifications repressor AD (de novo) Non- Ongoing Sz F, MF, SSW Severe DD with variant’) impairment frontal lobes Mutations impair Mid- specific in most, absent Hypotonia Scoliosis telencephalon >30 late LGS development and

325 refractory language, most development and

~15 in few - T, ambulatory postnatal neuronal M survival Yeung 2009 Autism Brunetti-Pierri 2011 Seltzer 2014 Pontrelli 2014 Bertossi 2014 GABRA1 1-15m DS/DS-like FS, TCS, HC, GSW, - UK Movement - Normal in most, One reported Ligand-gated ion MAE_like SE, IS, T photo- disorders atrophy in some death channel responsible AD (de novo) Other EE incl sensitivity Mild-severe DD (various) in some for inhibitory WS, EIEE Refractory in 50%,-F, neurotransmission >~23 Sz in most - B-S, hyps Mutations result in TCS, A, MJ, loss of function Epi4K 2013 Also causes F, aT As above Carvill 2014 non-EE Kodera 2016 epilepsy: FS, Johannesen 2016 mild GGE GABRB3 2-10 m WS IS, F,M, T Hyps, MF - DD in some - - Normal in most - Ligand-gated ion LGS channel responsible AD (de novo) Non-specific Refractory MF, hyps, Moderate- for inhibitory Sz in most - SSW profound DD neurotransmission 6 ADHD in some

326 GNAO1 0-2 m EIEE (5%) T B-S - N/A Movement - Normal early One reported G protein subunit in most WS disorder (most infancy death involved in cell AD (de novo) Non-specific Refractory Hyps or Severe- frequently signaling. Mutations Mid- Sz - IS, MF, later to profound DD choreoathetosis) Delayed impair adrenergic 9 child- later to T SSW, F B-S myelination, signaling and calcium hood +/- F thin corpus current, reducing Nakamura 2013 in callosum, cortical excitability Saitsu 2015 some progressive cerebral atrophy from later infancy GRIN1 0-18m Non-specific IS, F, M F, MF, - DD Movement - Cerebral Deaths in NMDA receptor ‘diffuse’ disorder (mixed, atrophy patients with subunit AD (de novo) Ongoing Sz, Severe- hyperkinetic Thin corpus homozygous (AR in 2 families) refractory As above profound DD including callosum mutations in 50%, Autistic oculogyric crisis- reported 16 controlled features like eye in 50% Behavioural movements, may Epi4K 2013 problems incl precede Sz onset) Ohba 2015 self-injurious Hypotonia or Lemke 2016 behaviour spastic quadriplegia Rett-like features

GRIN2B 1 m – 9 WS IS, M, F Hyps PN: NMDA UK Hypotonia - Normal in most - NMDA receptor y LGS inhibitors ‘Episodic subunit NR2B AD (de novo) Non-specific Refractory SSW, UK such as Mild-profound hyperextension’ Mutations reported to Sz - ES, T C, memantine DD Movement result in gain of 5 T, F, A disorder function (dystonia) Epi4K 2013 Lemke 2014 Smiegel 2016 HCN1 4-13 m DS-like FS, TCS, HC, N, GSW, - UK Ataxia in some - Normal - Hyperpolarization-

327 SE PSW, MF activated, cyclic AD (de novo) Mild-severe DD nucleotide–gated

Refractory GSW, PSW, Autism channel 6 Sz - TCS, A, MF Major Mutations can have F, M behavioural either a gain of Nava 2014 disturbance function or dominant negative effect on channel function IQSEC2 8 m- 4 IS aT, M, IS, Hyps, SSW, - UK Hypotonia - Normal or mild One reported Guanine nucleotide y Late-onset atA, GTCS GSW, PSW, Movement cerebral and death exchange factor for X-linked (affected spasms MF Moderate- disorder cerebellar the ARF family of GTP- males and LGS Refractory severe DD (stereotypies) atrophy and binding proteins, roles females reported) Non-specific Sz - M, As above Autistic Strabismus white matter include regulation of TCS, aT. ES, features Microcephaly and changes organelle formation 18 Also causes T, atA, F macrocephaly and vesicular non-EE reported transport Morleo 2008 generalized Shoubridge 2010 epilepsies Epi4K 2013 Mau-Them 2014 Gandomi 2014 Tzschacht 2015 Zerem 2016

328 most) Refractory photosensit profound DD disorder in some

10 Sz ive In-toeing in some

Torkamani 2014 Some Saitsu 2015 triggered Thiffault 2015 by stimuli Allen 2016 (eg De Kovel 2016 tripping, emotion) KCNQ2 Most EIEE (20%) T, F B-S, MF, F B: PHT, CBZ N/A Axial hypotonia - Basal ganglia Deaths Potassium channel in EOEE +/- spastic and thalamic reported ‘EE’ mutations can AD (de novo for week 1 Most settle MF PN: Moderate- quadriplegia hyperintensity result in loss or gain of EE, usually Also causes by 3 y - T, retigabine, profound DD Movement in infancy function, BFNS inherited in non-EE F, TCS, M ezogabine disorder mutations typically ‘benign’ epilepsy: Evolution (dystonia, non- loss of function epilepsies) BFNS to IS rare epileptic myoclonus) in >30 some Excess startle Weckhuysen 2012 and 2013 Kato 2013 Pisano 2015

BFNS: Singh 1998 KCNT1 0-6 m EIFMS (50%) F, T, IS MF, B-S PN: quinidine N/A or normal Hypotonia or Precocious Delayed Some deaths Potassium channel EIEE (single in most spasticity puberty in myelination reported Mutations produce AD (de novo) report), WS Refractory MF Acquired some Thin CC gain of function, (single Sz - F, IS Severe- microcephaly degree of gain of >30 report) (rare) profound DD function correlates with phenotype Barcia 2013 Also causes severity Heron 2013 Severe Milligan 2014 ADNFLE (mid-

329 Bearden 2014 childhood

Mikati 2015 onset) Chong 2016 KCTD7 0.5-3y PME M, GTC MF or GSW - Normal of mild Ataxia - Normal or mild Three Forms multimers, (most DD Pyramidal signs atrophy or reported affects potassium AR 1-2y) M, GTC, Microcephaly in posterior T2 deaths, signaling, glutamine atA, aT some WM survival into tranasport and 21 Severe DD hyperintensities adulthood is synaptic signalling Refractory (regression, reported Van Bogaert 2007 in many ultimately Kousi 2012 stabilizing) Van Bogaeart 2016 Moen 2016 KIAA2022 8m-18y ‘Generalised M, Ab, Sp PSW, hyps - Often DD Hypotonia Neonatal Normal in most One reported Role in neurite epilepsies’ Microcephaly in feeding death outgrowth X-linked (males Mild-severe DD some difficulties more severely M, GTC, Ab, (males more Dysmorphism affected) aT severe) Esotropia Neurobehaviou >30 Refractory ral disorders in most (autistic features,

330 Paciokowski 2013 myelination overlapping clinical

features PCDH19 4 m – 3 EFMR FS, F, SE N, F, GSW B: CLB, Normal-mild DD Unsteady gait in - Normal in most Deaths Cadherin family of y steroids, PHT, some reported cell-cell adhesion X-linked (male DS-like Sz-free in As above bromide Normal- molecules, involved in sparing unless ~30% moderate DD a variety of functions mosaic) Ongoing Sz PN: Autistic ‘Cellular interference clusters in Allopregnanol features moderateel’ >30 ~70%, less one proposed, in which frequent mixed population of Juberg 1971 over time - wild-type and mutant Ryan 1997 F, FS, M, A, cells required to Scheffer 2008 SE produce disease Dibbens 2008 (therefore affecting Depienne 2009 females and mosaic Marini 2010 and males) 2012 Higarushi 2013 Tan 2015 Lotte 2016 PIGA 0-9 m EME M, T, F, IS. B-S, hyps, N - N/A or DD in Axial hypotonia Polyhydramnios Delayed Death in early Biosynthesis of EIEE SE most Spastic Joint myelination childhood in glycosylphosphotidylin X-linked EOEE MF, UK quadriplegia contractures Thin CC some ositol anchors, WS Profound DD required to attach cell

331 Krawitz 2012

Nakamura 2014 PLCB1 2-10 m EIMFS F, T, IS, TCS, B-S, hyps, - DD Axial hypotonia - Normal or - Phospholipase enzyme EOEEWS SE MF +/- spastic Mild cortical involved in signal AR Non-specific Severe- quadriplegia atrophy transduction across Refractory UK profound DD Mildly cell membrane 4 Sz in most - hypoplastic F, T, TCS, SE corpus callosum Kurian 2010 Poduri 2012 Ngoh 2014 Schoonjans 2016 PNKP 1-6 m Non-specific F, FS UK - DD in some, UK Congenital - Mildly - DNA repair enzyme LGS in some microcephaly simplified gyral with both kinase and AR Refractory As above pattern phosphatase activity Sz in most - Severe DD Cerebellum Mutations result in 12 Also F, UK Hyperactive proportionate increased apoptosis associated behaviour to cerebrum and reduced cell Shen 2010 with non-EE proliferation Poulton 2012 epilepsy: Nakashima 2014 spinocerebell ar atrophy with

PURA 0-14 m LGS ES, M UK - DD Neonatal onset Strabismus Normal or - Roles in DNA in most Non-specific hypotonia, Multiple delayed transcriptional AD (de novo) Refractory MF, UK Severe DD in respiratory fractures myelination regulation and mRNA in some, most difficulties, poor trafficking in postnatal 7 not feeding brain development reported in ‘Myopathic’ facies Hunt 2014 others - M, Excess startle Lalani 2014 T, TCS, F, aT Recurrent aspiration Nystagmus

332 QARS Most EIMFS F, T, SE B-S, MF, - N/A Microcephaly - Progressive - Glutaminyl-tRNA in EIEE hyps (congenital in atrophy of synthetase, role in AR week 1 Non-specific Refractory Profound DD some, progressive cerebrum and protein translation. Sz UK in all) cerebellum Mutations lead to 7 Hypotonia +/- (vermis >> reduced neuronal spastic hemispheres) survival Zhang 2014 quadriplegia Delayed Kodera 2015 Episodes in myelination, Salvarinova 2015 childhood of reduced white agitation, matter, thin excitement, corpus callosum thrashing, poor sleep, excess sweating during which Sz are absent (and may  rhabdomyolysis) ROGDI 1m-4y Non-specific F, M, TCS, F, MF - DD or normal Spasticity Teeth Variety of non- Some deaths Function unknown (most atA anomalies specific in childhood AR in 1st Severe- abnormalities reported year) Refractory profound DD >30 for some time, then

333 Claes 2001 sensitive rate of

Harkin 2007 Also causes 9.3/1000 Freilich 2011 non-EE person years Catarino 2011 epilepsy: Brunklaus 2012 GEFS+, others Kim 2014 Cooper 2016 Ceulemans 2016

GEFS+: Escayg 2000, Wallace 2001 SCN2A 0-3 m Phenotypes: F, T, IS B-S, MF B: PHT, other N/A or normal Movement Severe Variety of non- Death in early Sodium channel in most neonatal/earl sodium in most disorders (mixed gastrointestinal specific findings childhood in subunit Nav1.2, AD (de novo for Early y infantile EE Refractory MF, N channel – dyskinesias in symptoms Signal change in some expressed on EE, usually child- presenting as Sz - F, IS, T, blockers Severe- intermediate white matter, excitatory neurons inherited in hood EIEE (10%) M profound DD in severity basal ganglia, Effect of mutations on ‘benign’ in EIMFS (25%) most, some neonatal/infantile thalami, channel function not epilepsies) some EOEE normal cases, mixed in brainstem well understood severe Atrophy Genotype-phenotype >30 neonatal/infantile (cortical +/- difference between cases, chorea in cerebellar)

Also causes non-EE epilepsy: BFNIS SCN8A 0-22 m EIMFS F, IS, T N, F, MF, B: PHT, other Normal or DD Movement - Normal Death in Sodium channel WS hyps sodium disorders (mixed, or atrophy, thin childhood in subunit Nav1.6,

334 AD (de novo) Non-specific Refractory channel Moderate- may precede Sz corpus some expressed on

Sz in most MF, SSW blockers profound DD onset) callosum, excitatory and >30 - F, T, C, M, Excess startle delayed inhibitory neurons Also causes atA, SE Hypotonia myelination Mutations produce Veeramah 2012 non-EE Spastic gain of function with Carvill 2013 epilepsy; quadriplegia in increase in persistent Epi4K consortium BFIS-like some sodium current and 2013 Ataxia in some incomplete channel Ohba 2014 inactivation Larsen 2015 Nav1.8 replaces Nav1.2 Wagnon 2015 as dominant sodium channel in excitatory BFIS-like epilepsy: neurons in infancy, Gardella 2016 which may explain Anand 2016 later age of Sz onset with SCN8A mutations than SCN2A SETBP1 0-1y in WS (in 25%), Sp, M, T, Hyps, B-S, - DD Hypotonia Neonatal Progressive Death before Ubiquitously most EME, EIEE, TCS MF Visual impairment feeding diffuse atrophy 10y in most expressed, function AD (de novo) non-specific Severe- Hearing problems Abnormal (respiratory unknown. Mutations 1/3 in Refractory profound DD impairment Dysmorphism myelination infections, though to exert gain- >30 (>70 cases neonat Sz in most – Other organ Malformative respiratory of-function or Schinzel-Giedion al Sp, M, T, abnormalities features in failure),

SIK1 0-4 m EME M, IS, T B-S, hyps Other: None N/A in most Repetitive and Scoliosis in Normal or mild Death in Salt-inducible kinase EIEE with infantile self-injurious some frontal lobe infancy in with roles in circadian AD (de novo) WS Refractory Hyps, UK spasms Severe DD in behaviours hypoplasia or neonatal rhythm, transcription Non-specific Sz - M, ES, responded to infant onset mildly onset cases of corticotrophins in

335 6 T, TCS, aT steroids cases (no simplified hypothalamus and

language, some gyration effects on MEF2C Hansen 2015 ambulant) pathway. Truncation Autism mutations result in loss of function. ACTH increases SIK1 activity. Further research required to determine if this is important in the mechanism of treatment response to steroids in infantile spasms. SLC1A2 0-1m EOEE M, T, IS MF, hyps - N/A Hypotonia or - Cerebral No reported Encodes a major spastic (no features atrophy and deaths glutamate transporter AD (de novo) Refractory Profound DD quadriplegia reported in abnormal EAAT2. Mutations Sz – more than one myelination impair glutamate multiple patient) uptake, increasing 3 types incl glutamate and

M, T, F, TCS resulting in Epi4K 2013 excitotoxicity Epi4K 2016

336 Thevenon 2014 in most dystonia) gingival functions Hardies 2015 (with Microcephaly hyperplasia) Klotz 2016 periods of Schossig 2016 relative stability or seizure freedom in childhood)- F, HC, TCS, M, fever- sensitivity in many SLC25A22 0-6 m EME M, F B-S, MF - N/A or DD Axial hypotonia Dysmorphism Normal or a Deaths Mitochondrial EIMFS Spastic variety of reported glutamate transporter AR WS Refractory MF, SSW, Profound DD quadriparesis findings Non-specific Sz - IS, M, T hyps Acquired including 9 microcephaly atrophy and Abnormal delayed Molinari 2005, ERG/VEP in some myelination 2009 Poduri 2013 Cohen 2014

337

SLC35A2 0-3m WS Sp, T, F Hyps - UK Hypotonia Dysmorphic Cerebral and - Encodes a UDP- EOEE WS cerebellar galactose transporter X-linked (mainly Severe- atrophy in most which is involved in females affected) profound DD Transferrin protein glycosylation. Refractory isoforms often Skewed X-inactivation 7 in most – (but not always) (WT allele expressed) Sp (seen at normal in the peripheral Ng 2013 some point tissues tested Kodera 2013 in all), F, T (postulated to explain Dorre 2015 absence of abnormal Kimizu 2016 transferrin isoforms and other organ abnormalities; also postulated that X- inactivtation patterns would be different in brain) SLC6A1 1-5.5 y EMA (4%) M, aT, A GSW, PSW, B: KD DD Tremor or ataxia - Normal in most - Voltage-dependent some in some GABA transporter AD (de novo) photo- Mild-severe DD responsible for sensitive synaptic GABA

SMC1A 1-17m Focal F, M, Sp, F, MF (IEDs - Many DD (but Hypotonia or Facial Variety of non- No deaths Protein has a role in epilepsies TCS predomina may be v mild) spastic dysmorphisms specific reported chromosome cohesion X-linked (mainly Other ntly quadriplegia (not always features frameshift, some posterior) synophrys as is missense Refractory Mod-severe DD seen in CdL)

338 mutations), Sz – mainly (non-verbal) Small hands and

female affected Milder F feet phenotypes Prenatal growth 6 in males and Seizures in restriction females with clusters in Goldstein 2015 missense and some Jansen 2016 in-frame del Some similar muts – features to CdL, These are but not full associated hand and with Cornlia sometimes de Lange none present syndrome. Epilepsy, where present is usually FS or treatment- responsive childhood onset focal Sz

339 7 hood T microcephaly cerebral, effect lead to altered

onset brainstem and clustering of sodium Saitsu 2010 cases cerebellar channels at axon Hamdan 2012 atrophy initial segment and Writzl 2012 impaired integrity of Nonoda 2013 myelinated axons. Tohyama 2015 Haploinsufficiency thought not to cause EE STXBP1 0-12y EIEE (30%) T, F, IS, M, MF, F, B-S, PN: Alpha N/A or DD Movement - Normal (~50%) - Synaptic protein (most EOEE FS, TCS hyps helical disorder (esp Cortical atrophy Mutations disrupt AD (de novo) 0-3m) WS (2%) protein: Severe- tremor - ‘figure of or hypoplasia synaptic transmission DS Seizure free MF, protein profound DD in 8 head tremor’, (mainly frontal) >30 EME in >1/3. unusual interaction most stereotypies) Thin +/- Ongoing back- inhibitors Autistic Hypotonia dysmorphic CC Saitsu 2008 seizure sin ground fast features in Spastic Hypo- or Deprez 2010 many -may without some quadriplegia delayed Otsuka 2010 settle then IEDs Ataxia myelination Mignot 2011 recur in Normal head Milh 2011 later growth Carvill 2014 infancy Di Meglio 2015 Stamberger 2016

340 Carvill 2013 Dyment 2014

Mignot 2016 SZT2 2m-4y Non-specific F, T, atA MF, - DD Hypotonia, Dysmorphism Thick corpus One death Function unknown bilaterally reflexes may be Scoliosis in callosum reported AR (3/4 at synchronou Profound DD reduced some PVNH in one 2m) s SW patient 4

Basei-Vanagaite 2013 Venkatesan 2016 TBC1D24 2-3 m EIFMS F, SE, M, MF, UK - DD in some DOORS syndrome DOORS Subtle cortical Some GTPase-activating in most Other TCS, A Cerebellar signs syndrome thickening, reported protein involved in AR focal/multifoc As above Mild-profound Hearing Nail anomalies progressive deaths cycling of synaptic al infantile Refractory DD impairment Bony anomalies signal change vesicles, membrane >30 epilepsy Sz - F, M and atrophy in recycling, actin Other cerebellar remodelling and Corbett 2010 infantile ansiform neurite development Guven 2012 myoclonic lobules Mutations mainly Milh 2013 epilepsies affect Afawi 2013 phosphoinositide- Campeau 2014 Also causes binding pocket, inhibit Balestrini 2016 non-EE lipid binding and Fischer 2016

Gene Age Sz Epilepsy Sz at onset EEG at Notable Development Other features – Other features Magnetic Survival Protein function and onset syndromes onset treatments at onset neurologic – non- resonance mechanistic Inheritance (% of cases of Sz neurologic brain imaging implications this evolution EEG Development Cases with severe syndrome evolution evolution epilepsy reported where known) References epilepsy: impair synaptic vesicle FIME trafficking. FIME: Falace 2010 UBA5 2-9m WS IS, M Hyps, other - DD in most Dystonia Failure to Atrophy Death in Role in post- Non-specific Hypotonia  thrive, Delayed approximatel translational protein AR Refractory Severe- spastic progressive myelination, y half modification Sz where profound DD quadriparesis growth failure white matter (‘ufmylation’) 11 reported – Acquired hyperintensities M, GTC, F microcephaly Thin corpus Muuna 2016 Jittery and callosum Colin 2016 irritable as neonates WDR45 3-6m WS IS, F Hyps, MF - DD in most Hypotonia, spastic - Cerebral - Role in autophagy 341 Non-specific quadriplegia, atrophy X-linked (mainly Refractory Severe- dystonia in some Delayed male) Sz profound DD myelination Also (sometimes Iron deposition 5 (1 female) associated after a noted in two with BPAN in period of males at 4y and Abidi 2014 females, with seizure 5y Xixis 2015 childhood freedom) Nakashima 2016 onset seizures in many

WWOX 0-5m WS IS, F, T, TCS, Hyps, MF - DD in some ‘EE’ phenotype: FTT Normal or Death in WW-domain in most LGS M Spasticity (often cerebral (but infancy/ early containing AR Other MF, SSW Moderate- early onset – 1- not cerebellar) childhood in oxidoreductase, Up to 2 focal/multifoc Refractory profound DD 3mo) atrophy, most with involved in a variety of 18 y in al Sz - T, F, M (profound DD in Hypokinesia periventricular ‘EE’ pre- and postnatal some Focal WS all with ‘EE’ Acquired whie matter phenotype cellular processes Mallaret 2014 (later LGS in some phenotype) microcephaly loss, thin/ (unclear which are Abdel-Salam onset Retinal hypoplastic relevant to epilepsy 2014 for abnormalities or corpus callosum pathogenesis) Mignot 2015 ‘SCAR’ optic atrophy in Ben-Salam 2015 phenot many Null mutations Tabarki 2015 ype) produce ‘EE’

Abbreviations:

Epilepsy syndromes: BFNIS benign familial neonatal-infantile Sz, BFNS benign familial neonatal Sz, CAE childhood A epilepsy, DS Dravet syndrome, EAS epilepsy aphasia spectrum, EFMR epilepsy limited to females with mental retardation, EIEE early infantile epileptic encephalopathy (Ohtahara syndrome), EIMFS Epilepsy of infancy with migrating F Sz, EMA epilepsy with M aT Sz, EME early M epileptic encephalopathy, EO-AE

342 early onset A epilepsy, EOEE early onset epileptic encephalopathy, FIME familial infantile M epilepsy, FS febrile Sz, GEFS+ generalized epilepsy with febrile Sz plus, GGE genetic generalized epilepsy, LGS Lennox-

Gastaut syndrome, WS West syndrome

Seizure types: A absence, A(EM) absence with eyelid myoclonia, aT atonic, atA atypical absence, ES epileptic spasms, F focal, FS febrile seizures, HC = hemiconvulsive, IS infantile spasms, M myoclonic, SE status epilepticus, T tonic, TCS tonic clonic seizure

EEG features: B-S burst-suppression, F focal, GSW generalized spike-wave, MF multifocal, PSW polyspike-wave, SSW slow spike-wave

Notable treatments: B beneficial, BDZ benzodiazepine, CBZ carbamazepine, E exacerbating, KD ketogenic diet, LTG lamotrigine, OXC oxcarbazepine, PHT phenytoin, PN potential novel, TPM topiramate, VPA valproate, ZNS zonisamide

Other: ACTH adrenocorticotropic hormone, AD autosomal dominant, AED antiepileptic drug, AR autosomal recessive, d day, DOORS syndrome deafness, onychodystrophy, osteodystrophy, retardation and seizure syndrome, m month, Sz seizures, y years

Appendix B: Study clinical assessment case report form

IEE study CRF 27022013 343 Study ID

Initials Demographics Study ID Surname First name Gender DOB State of birth Street address Suburb Postcode State Phone number Living? COD DOD RCH UR

Visit information Visit number Visit date

Ascertainment information Method Site Referring doctor Treating doctor (if different) Previous enrolment in 20114A?

Family information *draw family tree on reverse* Consanguineous? Parents Siblings Miscarriage/still births Epilepsy Febrile convulsions Intellectual disability Autism spectrum disorder Migraine Psychiatric conditions Other

IEE study CRF 27022013 344 Study ID

Initials Perinatal and non-neurologic medical history Pregnancy

USS abN? Movement abN? Liquor volume? Other Perinatal/neonatal

Gestation Problems with delivery APGARs 1min 5mins 10mins Neonatal seizures? Ventilation required? Established feeding normally? Hypoglycaemia? Hyperbilirubinaemia? Other Other medical

Other organ abN? Major illness – neurologic Major illness – non-neurologic Supplemental feeding required Other

IEE study CRF 27022013 345 Study ID

Initials Epilepsy history Age at onset Seizures ongoing? Current frequency Age at offset On/off treatment? Seizure types

Number of types Number of current types *if >5 types, add information overleaf* Type #1

Name Onset Description Frequency current maximum Time to treatment Ceased with first medication? Ceased with any treatment? Offset Type #2

Name Onset Description Frequency current maximum Time to treatment Ceased with first medication? Ceased with any treatment? 2.1.1 Offset

Type #3

Name Onset Description Frequency current maximum Time to treatment Ceased with first medication? Ceased with any treatment? Offset

IEE study CRF 27022013 346 Study ID

Initials Type #4

Name Onset Description Frequency current maximum Time to treatment Ceased with first medication? Ceased with any treatment? Offset Type #5

Name Onset Description Frequency current maximum Time to treatment Ceased with first medication? Ceased with any treatment? Offset Other seizure details

Neonatal seizures Febrile convulsions Status epilepticus Clusters Timing Triggers Seizure free periods Other

IEE study CRF 27022013 347 Study ID

Initials EEG Number Sites Age at first EEG Seizures recorded? If known: EEG Age Site Detail

Seizure videos available? Seizure type Home video EEG video

Other videos available? (eg of movement disorder, of gait) *Give detail*

IEE study CRF 27022013 348 Study ID

Initials Treatments Current Medication used

Drug Order used Dose adequate Effect SEs ACZ CBZ CLB CZP DZP ESM FLB GBP LCS LEV LTG NZP OXC PB PHT SLT TPM VGB VPA ZNS PNL IVIg B6 P5P Folinic acid Biotin Other

Combinations used Most effective medication/combination

IEE study CRF 27022013 349 Study ID

Initials Non-pharmacologic treatment Ketogenic diet

Used? Date commenced Effect? Ceased? Reason VNS

Used? Date implanted Effect? Ceased? Reason

Surgery

Performed? Date ECoG intraoperative monitoring Procedure performed Outcome Other Emergency management

Emergency anticonvulsants required? Use

Prolonged seizure

Cluster seizures

Cardiorespiratory compromise

Other Type

MDZ

DZP

CZP

Other

IEE study CRF 27022013 350 Study ID

Initials

Frequency of use Effect Subsequent emergency AED use by ambulance/hospital required?

Neurodevelopmental history Age-appropriate? Delay prior to seizures? Age delay first noted? Plateau/regression?

Number of episodes

Age

Trigger

Skills lost

Skills Parental estimate of functional age Visually responsive? Smiles responsively? Motor

Skill Acquired? Age

Roll

Sit

Walk

Current best skill Language

Skill Acquired Age

Coo

Babble

Single words

Responds to voice

Responds to name

Follows single part commands

IEE study CRF 27022013 351 Study ID

Initials

Current best skill Other neurodevelopmental Concerns re social skills Behavioural difficulties Neuropsych testing performed?

Test

Site

Age

FSIQ? ASD diagnosis Psychiatric diagnosis Other

Neurologic Handedness

Left/right/ambidextrous

Early?

FHx sinistrality?

Hypotonia Paresis Spasticity Movement disorder Ataxia PNS abN Non-epileptic paroxysmal disorder

Migraine

Hyperrekplexia

Breath holding

Cataplexy

Syncope

Pseudoseizure

Parasomnia

Other

IEE study CRF 27022013 352 Study ID

Initials

Head circumference Autonomic symptoms Strabismus Vision concerns Hearing concerns? Other

Cause Known? Age at diagnosis Method of diagnosis Site of diagnosis Other Investigations performed

Brain imaging Blood tests Urine tests CSF sampling Gene testing Biopsies Woods lamp Ophthal r/v Hearing assessment Brain histology Post-mortem Other Additional tests planned

IEE study CRF 27022013 353 Study ID

Initials Services involved

Neurologist Neonatologist Paediatrician Psych Palliative care Other medical Early intervention PT OT S/P Dietician S/W N/psych Other Consults re diagnosis

Ophthalmology Metabolics Genetics Other

Medical records Site UR Clinical EEG MRI Other Ix Consent Records Records to obtain requested obtained

RCH MMC Austin Mercy RWH

Examination of patient Size

Length Dysmorphology

Facial

Other

IEE study CRF 27022013 354 Study ID

Initials

Neurocutaneous stigmata Organomegaly Seizures seen during assessment? Development Neurologic

Hypotonia

Spasticity

Hemiparesis

Handedness

Movement disorder

Ataxia

PNS abN

Autonomic abN

Strabismus

Vision

Hearing

Other Other

IEE study CRF 27022013 355

356

Appendix C: Known and candidate genes included in the molecular inversion probe-based multigene panels

Number Version of of MIPS Genes in target patients target tested ADAM22, ADAM23, ARX, CACNA2D1, CDKL5, CPLX1, DLGAP1, 7 T1 DLGAP2, DLGAP3, EPHA6, GABRA1, GABRG2, GNAI1, GRIN2A, KCNQ2, MAPK8, OTX1, PCDH19, SCN1A, SCN1B, SCN2A, SEMA3A, SH3GL2, SLC1A1, SLC1A3, SLC2A1, SMARCA2, SPTAN1, STXBP1, (T1 and T2 SV2A, SV2B, SYT2 AKT3, APBA1, APBA2, ARHGEF9, ATF7IP2, ATP1A2, CACNB4, CHD2, performed T2 CHD5, CHRNA7, CLVS2, EPHB2, EPN1, ERBB4, FOXG1, HNRNPU, separately) KCNQ3, MBD5, MECP2, MEF2C,NUP188, PLCB1, PNKP, PNPO, PRICKLE1, SCN8A, SCN9A, SEMA3E, SLC25A22, STX1A, SYN1, SYNGAP1, UBE3A, ZNF532 AKT3, ALG13, ARHGEF9, ARX, ATP6V0C2, CACNB4, CDKL5, CHD2, CHD5, CLSTN1, EEF1A2, FOXG1, GABRA1, GABRB3, GABRD, T1T2 GABRG2, GRIN2A, GRIN2B, HNRNPU, KCNQ2, KCNQ3, KCNT1, 1 MBD5, MECP2, MEF2C, NKAIN1, NKAIN2, NKAIN3, NKAIN4, NOL11, PCDH19, PLCB1, PNKP, PNPO, SCN1A, SCN1B, SCN2A, SCN8A, SCL1A1, SLC1A3, SLC25A22, SLC2A1, SPTAN1, STXBP1, SYN1, SYNGAP1, SYT2, UBE3A AKT3, ARHGEF9, ARX, ATP6V0C2, CACNB4, CDKL5, CHD2, CHD5, CLSTN1, FOXG1, GABRA1, GABRD, GABRG2, GRIN2A, GRIN2B, T1T2kv1 1 HNRNPU, KCNQ2, KCNQ3, KCNT1, MBD5, MECP2, MEF2C, NKAIN1, NKAIN2, NKAIN3, NKAIN4, NOL11, PCDH19, PLCB1, PNKP, PNPO, SCN1A, SCN1B, SCN2A, SCN8A, SCL1A1, SLC1A3, SLC25A22, SLC2A1, SPTAN1, STXBP1, SYN1, SYNGAP1, SYT2, UBE3A AKT3, ALG13, ARHGEF9, ARX, ATP6V0C2, CACNB4, CDKL5, CHD2, CHD5, CLSTN1, EEF1A2, FOXG1, GABRA1, GABRB3, GABRD, T1T2kv2 GABRG2, GRIN2A, GRIN2B, HNRNPU, KCNQ2, KCNQ3, KCNT1, 2 MBD5, MECP2, MEF2C, NKAIN1, NKAIN2, NKAIN3, NKAIN4, NOL11, PCDH19, PLCB1, PNKP, PNPO, SCN1A, SCN1B, SCN2A, SCN8A, SCL1A1, SLC1A3, SLC25A22, SLC2A1, SPTAN1, STXBP1, SYN1, SYNGAP1, SYT2, UBE3A ALG13, ARHGEF9, ARX, CDKL5, CHD2, CUX2, DEPDC5, DNM1, EEF1A2, FOXG1, GABRA1, GABRB1, GABRB3, GABRG2, GNAO1, T1T2kv3 26 GRIN2A, GRIN2B, HNRNPU, KCNH5, KCNQ2, KCNQ3, KCNT1, MBD5, MECP2, MEF2C, PCDH19, PLCB1, PNKP, PNPO, SCN1A, SCN1B, SCN2A, SCN8A, SLC25A22, SLC2A1, STXBP1, SYNGAP1, TCF4, UBE3A ALG13, ARHGEF9, ARX, CACNA1A, CDKL5, CHD2, CUX2, DEPDC5, DNM1, EEF1A2, FOXG1, GABRA1, GABRB1, GABRB3, GABRG2, T1T2kv4 7 GNAO1, GRIN2A, GRIN2B, HNRNPU, IQSEC2, KCNH5, KCNQ2, KCNQ3, KCNT1, MBD5, MECP2, MEF2C, PCDH19, PLCB1, PNKP, PNPO, SCN1A, SCN1B, SCN2A, SCN8A, SLC1A2, SLC25A22, SLC2A1, SLC6A1, STXBP1, SYNGAP1, TCF4, UBE3A, WDR45 NB Six infants with structural or metabolic aetiologies in this cohort had MIPS testing and are listed in this table

357

358

Appendix D: Genes included in targeted analysis of whole exome sequencing

ILAE Gene Inheritanc OMIM OMIM gene Condition name classification name^ e pattern phenotype* group

AARS AR 601065 616339 EIEE29 Genetic

AARS2 AR 612035 614096 Combined oxidative phosphorylation deficiency 8 Metabolic

ABAT AR 137150 613163 GABA-transaminase deficiency Metabolic

ACOX1 AR 609751 264470 Peroxisomal acyl-CoA oxidase deficiency Metabolic

ADAR AR 146920 615010 Aicardi-Goutieres ysndorme 6 Genetic

ADSL AR 608222 103050 Adenylosuccinase deficiency Metabolic

AIMP1 AR 603065 260600 Hypomyelinating leukodystrophy 3 Genetic

359

AKT1 AD 164730 176920 Proteus syndrome Structural

AKT3 AD 611223 615937 Megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome 2 Structural

ALDH5A1 AR 610045 271980 Succinic semialdehyde dehydrogenase deficiency Metabolic

ALDH7A1 AR 107323 266100 Pyridoxine-dependent epilepsy Metabolic

ALG1 AR 605907 608540 Congenital disorder of glycosylation, type 1K Metabolic

ALG11 AR 613666 613661 Congenital disorder of glycosylation, type 1P Metabolic

ALG12 AR 607144 607143 Congenital disorder of glycosylation, type 1G Metabolic

ALG13 X-linked 300776 300884 EIEE36 Metabolic

ALG2 AR 607905 607906 Congenital disorder of glycosylation, type 1I Metabolic

ALG3 AR 608750 601110 Congenital disorder of glycosylation, type 1D Metabolic

ALG6 AR 604566 603147 Congenital disorder of glycosylation, type 1C Metabolic

ALG8 AR 608103 608104 Congenital disorder of glycosylation, type 1H Metabolic

ALG9 AR 606941 608776 Congenital disorder of glycosylation, type 1L Metabolic

ILAE Gene Inheritanc OMIM OMIM gene Condition name classification name^ e pattern phenotype* group

ALPL AR 171760 241500 Hypophosphatasia (infantile) Metabolic

AMPD2 AR 102771 615809 Pontocerebellar hypoplasia type 9 Structural

AMT AR 238310 605899 Glycine encephalopathy Metabolic

ARFGEF2 AR 605371 608097 Periventricular heterotopia with microcephaly Structural

ARHGEF15 AD 608504 ** SINGLE Genetic CASE

ARHGEF9 X-linked 300429 300607 EIEE8 Genetic

ARID1B AD 614556 135900 Coffin-Siris syndrome Genetic

ARX X-linked 300382 308350 EIEE1, X-linked lissencephaly with abnormal genitalia Genetic AND Structural

360

ASPA AR 608034 271900 Canavan disease Metabolic

ATP5A1 AR 164360 multiple Combined oxidative phosphorylation deficiency 22 (616045), Metabolic phenotype IDs Mitochondrial complex deficiency nuclear type 4 (615228)

ATP6AP2 X-linked 300556 300423 Mental retardation, X-linked, syndromic, Hedera type Genetic

ATP7A X-linked 300011 309400 Menkes disease Metabolic

ATRX X-linked 300032 multiple Mental retardation-hypotonic facies syndrome X-linked (309580), Alpha- Genetic phenotype IDs thalassemia/mental retardation syndrome (301040)

BCKDHA AR 608348 248600 Maple syrup urine disease, type 1A Metabolic

BCKDHB AR 248611 248600 Maple syrup urine disease, type 1B Metabolic

BCS1L AR 603647 multiple Leigh syndrome (256000), Mitochondrial complex III deficiency nuclear Metabolic phenotype IDs type 1

BOLA3 AR 613183 614299 Mulitple mitochondrial dysfunctions syndrome 2 with hyperglycinemia Metabolic

BRAF AD 164757 115150 Cardiofaciocutaneous syndrome Structural

BRAT1 AR 614506 614498 Rigidity and multifocal seizure syndrome, lethal neonatal Genetic

ILAE Gene Inheritanc OMIM OMIM gene Condition name classification name^ e pattern phenotype* group

BTD AR 609019 253260 Biotinidase deficiency Metabolic

C12ORF57 AR 615140 218340 Temtamy syndrome Structural

CACNA1A AD 601011 617106 EIEE42 Genetic

CACNA2D2 AR 607082 SINGLE Genetic FAMILY

CASK X-linked 300172 300749 Mental retardation and microcephaly with pontine and cerebellar Structural hypoplasia

CC2D2A AR 612013 612285 Joubert syndrome 9 Structural

CCND2 AD 123833 615938 Megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome 3 Structural

CDKL5 X-linked 300203 300672 EIEE2 Genetic

361

CHD2 AD 602119 615369 Epileptic encephalopathy, childhood-onset Genetic

CHRNA2 AD 118502 610353 Nocturnal frontal lobe epilepsy type 4 Genetic

CHRNA4 AD 118504 600513 Nocturnal frontal lobe epilepsy type 1 Genetic

CHRNB2 AD 118507 605375 Nocturnal frontal lobe epilepsy type 3 Genetic

CLCN4 X-linked 302910 300114 Mental retardation, X-linked 49 Genetic

CLN3 AR 607042 204200 Batten disease (Neuronal ceroid lipofuscinosis type 3) Metabolic

CNTNAP2 AR 604569 610042 Cortical dysplasia-focal epilepsy syndrome Structural

COG4 AR 606976 613489 Congenital disorder of glycosylation type 2J Metabolic

COG6 AR 606977 614576 Congenital disorder of glycosylation type 2L Metabolic

COG7 AR 606978 608779 Congenital disorder of glycosylation type 2E Metabolic

COG8 AR 606979 611182 Congenital disorder of glycosylation type 2H Metabolic

COL18A1 AR 120328 267750 Knobloch syndrome type 1 Structural

COL4A1 AD 120130 175780 Porencephaly 1 Structural

ILAE Gene Inheritanc OMIM OMIM gene Condition name classification name^ e pattern phenotype* group

COL4A2 AD 120090 614483 Porencephaly 2 Structural

COQ2 AR 609825 607426 Coenzyme Q10 deficiency, primary 1 Metabolic

COQ4 AR 612898 616276 Coenzyme Q10 deficiency, primary 7 Metabolic

COQ6 AR 614647 614650 Coenzyme Q10 deficiency, primary 6 Metabolic

COQ9 AR 612837 614654 Coenzyme Q10 deficiency, primary 5 Metabolic

COX10 AR 602125 multiple Leigh syndrome (256000), Mitochondrial complex IV deficiency (220110) Metabolic phenotype IDs

COX15 AR 603646 multiple Leigh syndrome (256000), cardioencephalopmyopathy fatal infantile Metabolic phenotype IDs (615119)

CSTB AR 601145 254800 Progressive myoclonic epilepsy 1A (Unverricht and Lundborg) Genetic

362

CTSD AR 116840 610127 Neuronal ceroid lipofuscinosis 10 (congenital form) Metabolic

D2HGDH AR 609186 600721 D-2-hydrogyglutaric aciduria Metabolic

DBT AR 248610 248600 Maple syrup urine disease type II Metabolic

DCX X-linked 300121 300067 Lissencephaly X-linked, Subcortical band heterotypia X-linked Structural

DEPDC5 AD 614191 604364 Familial focal epilepsy with variable foci 1 Structural AND Genetic

DHCR24 AR 606418 602398 Desmosterolosis Structural

DNAJC6 AR 608375 615528 Parkinson disease 19B, early-onset Genetic

DNM1 AD 602377 616346 EIEE31 Genetic

DOCK7 AR 615730 615859 EIEE23 Genetic

DOLK AR 610746 610768 Congenital disorder of glycosylation, type 1M Metabolic

DPAGT1 AR 191350 608093 Congenital disorder of glycosylation, type 1J Metabolic

DPM1 AR 603503 608799 Congenital disorder of glycosylation, type 1E Metabolic

ILAE Gene Inheritanc OMIM OMIM gene Condition name classification name^ e pattern phenotype* group

DPM2 AR 603564 615042 Congenital disorder of glycosylation, type 1U Metabolic

DYNC1H1 AD 600112 614563 Mental retardation, autosomal dominant 13 Structural

EARS2 AR 612799 614924 Combined oxidative phosphorylation deficiency 12 Metabolic

EEF1A2 AD 602959 616409 EIEE33 Genetic

EFHC1 AR 608815 SINGLE Genetic FAMILY

EIF2B1 AR 606686 603896 Leukoencephalopathy with vanishing white matter Structural

EIF2B2 AR 606454 603896 Leukoencephalopathy with vanishing white matter Structural

EIF2B3 AR 606273 603896 Leukoencephalopathy with vanishing white matter Structural

EIF2B4 AR 606687 603896 Leukoencephalopathy with vanishing white matter Structural

363

EIF2B5 AR 603945 603896 Leukoencephalopathy with vanishing white matter Structural

EMX2 AD 600035 269160 Schizencephaly Structural

EPG5 AR 615068 242840 Vici syndrome Structural

EPM2A AR 607566 254780 Progressive myoclonic epilepsy 2B (Lafora) Metabolic

ETHE1 AR 608451 602473 Ethylmalonic encephalopahty Metabolic

EXOSC3 AR 606489 614678 Pontocerebellar hypoplasia type 1B Structural

FARS2 AR 611592 614946 Combined oxidative phosphorylation deficiency 14 Metabolic

FASTKD2 AR 612322 220110 Mitochondrial complex IV deficiency Metabolic

FBXL4 AR 605654 615471 Mitochondrial DNA depletion syndrome 13 Metabolic

FGFR3 AD 134934 multiple Achondroplasia (100800), hypochrondroplasia (146000), Muenke Genetic phenotype IDs syndrome (602849)

FH AR 136850 606812 Fumarase deficiency Metabolic

FIG4 AR 609390 612691 Polymicrogyria, bilateral temporooccipital Structural

ILAE Gene Inheritanc OMIM OMIM gene Condition name classification name^ e pattern phenotype* group

FKRP AR 606596 613153 Muscular dystrophy-dystroglycanopathy (congenital with brain and eye Structural anomalies), Type A5

FKTN AR 607440 253800 Muscular dystrophy-dystroglycanopathy (congenital with brain and eye Structural anomalies), Type A4

FLNA X-linked 300017 300049 Periventricular nodular heterotopia Structural

FOLR1 AR 136430 613068 Cerebral folate transport deficiency Metabolic

FOXG1 AD 164874 613454 Rett syndrome congenital variant Genetic

FOXRED1 AR 613622 multiple Leigh syndrome (256000), Mitochondrial complex I deficiency (252010) Metabolic phenotype IDs

FUCA1 AR 612280 230000 Fucosidosis Metabolic

GABRA1 AD 137160 615744 EIEE19 Genetic

364

GABRB3 AD 137192 617113 EIEE43 Genetic

GABRG2 AD 137164 611277 Generalised epilepsy with febrile seizures plus type 3 Genetic

GALC AR 606890 245200 Krabbe disease Metabolic

GAMT AR 601240 612736 Cerebral creatine deficiency syndrome 2 Metabolic

GBA AR 606463 multiple Gaucher disease (perinatal lethal 608013, type II 230900) Metabolic phenotype IDs

GCH1 AR 600225 233910 Hyperphenylalaninemia, BH4-deficiency B Metabolic

GCSH AR 238330 605899 Glycine encephalopathy Metabolic

GFAP AD 137780 203450 Alexander disease Metabolic

GFM1 AR 606639 609060 Combined oxidative phosphorylation deficiency 1 Metabolic

GLB1 AR 611458 230600 GM1 gangliosidosis Metabolic

GLDC AR 238300 605899 Glycine encephalopathy Metabolic

GLI3 AD 165240 146510 Pallister-Hall syndrome Structural

ILAE Gene Inheritanc OMIM OMIM gene Condition name classification name^ e pattern phenotype* group

GLUD1 AD 138130 606762 Hyperinsulinism-hyperammonemia syndrome Metabolic

GLUL AR 138290 610015 Congenital glutamine deficiency Metabolic

GM2A AR 613109 272750 GM2-gangliosidosis AB variant Metabolic

GNAO1 AD 139311 615473 EIEE17 Genetic

GNAQ AD 600998 185300 Sturge-Weber syndrome Structural

GOSR2 AR 604027 614018 Progressive myoclonic epilepsy 6 Genetic

GPHN AR 603930 615501 Molybdenum cofactor deficiency C Metabolic

GPR56 AR 604110 606854 Polymicrogyria, bilateral frontoparietal Structural

GRIN1 AD 138249 614254 Mental retardation, autosomal dominant 8 Genetic

365 GRIN2A AD 138253 245570 Epilepsy, focal, with speech disorder and with or without mental Genetic retardation

GRIN2B AD 138252 616139 EIEE27 Genetic

GTPBP3 AR 608536 616198 Combined oxidative phosphorylation deficiency 23 Metabolic

HAX1 AD 605998 610738 Neutropenia, severe congenital 3 autosomal recessive Genetic

HCCS X-linked 300056 309801 Linear skin defects with multiple congenital anomalies Structural

HCFC1 X-linked 300019 309541 Methylmalonic acidemia and homocysteinemia cblX type Metabolic

HCN1 AD 602780 615871 EIEE24 Genetic

HEPACAM AR 611642 613925 Megalencephalic leukoencephalopathy with subcortical cysts 2A Structural

HEXA AR 606869 272800 Tay-Sachs disease Metabolic

HEXB AR 606873 268800 Sandhoff disease Metabolic

HLCS AR 609018 253270 Holocarboxylase synthetase deficiency Metabolic

HNRNPH1 AD 601035 SINGLE CASE Genetic

HNRNPU AD 602869 de Kovel et al Genetic

ILAE Gene Inheritanc OMIM OMIM gene Condition name classification name^ e pattern phenotype* group

HOXA1 AR 142955 601536 Athabaskan brainstem dysgenesis syndrome, Bosley-Salih-Alorainy Genetic syndrome

HPRT1 X-linked 308000 300322 Lesch-Nyhan syndrome Metabolic

HRAS AD 190020 218040 Costello syndrome Structural?

HSD17B4 AR 601860 261515 D-bifunctional protein deficiency Metabolic

HSPD1 AR 118190 612233 Hypomyelinating leukodystrophy 4 Structural

IER3IP1 AR 609382 614231 Microcephaly, epilepsy and diabetes syndrome Metabolic?

IFIH1 AD 606951 615846 Aicardi-Goutieres syndrome 7 Genetic

IKBKG X-linked 300248 308300 Incontinential pigmenti Structural?

ISPD AR 614631 614643 Muscular dystrophy-dystroglycanopathy (congenital with brain and eye Structural 366 anomalies, Type A7

KCNA2 AD 176262 616366 EIEE32 Genetic

KCNB1 AD 600397 616056 EIEE26 Genetic

KCNC1 AD 176258 616187 Progressive myoclonic epilepsy 7 Genetic

KCND2 AD 605410 SINGLE Genetic FAMILY

KCNH5 AD 605716 SINGLE CASE Genetic

KCNJ10 AD 602208 612780 SESAME syndrome (seizure, sensorineural deafness, ataxia, mental Genetic retardation and electrolyte imbalance)

KCNJ11 AD 600937 606176 DEND syndrome (developmental delay, epilepsy and neonatal diabetes) Metabolic

KCNQ2 AD 602235 613720 EIEE7 Genetic

KCNQ3 AD 602232 121201 Benign neonatal seizures type 2 Genetic

KCNT1 AD 608167 614959 EIEE14 Genetic

KCTD7 AR 611725 611726 Progressive myoclonic epilepsy 3 Genetic

ILAE Gene Inheritanc OMIM OMIM gene Condition name classification name^ e pattern phenotype* group

KIF1A AD 601255 614255 Mental retardation, autosomal dominant 9 Genetic

KIF1BP AR 609367 609460 Goldberg-Shprintzen syndrome Structural

KIF2A AD 602591 615411 Cortical dysplasia, complex, with other brain malformations 3 Structural

KIF5C AD 604593 615282 Cortical dysplasia, complex, with other brain malformations 2 Structural

KPNA7 AR 614107 SINGLE Structural FAMILY

KRAS AD 190070 multiple Cardiofaciocutaneous syndrome 2 (615278), Noonan syndrome 3 (609942) Structural phenotype IDs

LARGE1 AR 603590 613154 Muscular dystrophy-dystroglycanopathy (congenital with brain and eye Structural anomalies, Type A6

LIAS AR 607031 614462 Hyperglycinemia, lactic acidosis and seizures Metabolic

367

LMNB2 AR 150341 SINGLE Genetic

FAMILY

MAGI2 AD 606382 only deletions Genetic pathogenic?

MANBA AR 609489 248510 Mannosidosis, beta Metabolic

MAP2K1 AD 176872 615279 Cardiofaciocutaneous syndrome 3 Structural

MAP2K2 AD 601263 615280 Cardiofaciocutaneous syndrome 4 Structural

MBD5 AD 611472 156200 Mental retardation, autosomal dominant 1 Genetic

MECP2 X-linked 300005 312750 Rett syndrome Genetic

MED12 X-linked 300188 multiple Lujan-Fryns syndorme (309520), Opitz-Kaveggia syndrome (305450) Structural phenotype IDs

MED17 AR 603810 613668 Microcephaly, postnatal progressive, with seizures and brain atrophy Genetic

MEF2C AD 600662 613443 Mental retardation, stereotypic movements, epilepsy and/or cerebral Genetic malformations

ILAE Gene Inheritanc OMIM OMIM gene Condition name classification name^ e pattern phenotype* group

MLC1 AR 605908 604004 Megalencephalic leukoencephalopathy with subcortical cysts Structural

MMADHC AR 611935 277410 Methylmalonic acidemia and homocysteinemia cblD type Metabolic

MMACHC AR 609831 277400 Methylmalonic acidemia and homocysteinemia cblC type Metabolic

MOCS1 AR 603707 252150 Molybdenum cofactor deficiency A Metabolic

MOCS2 AR 603708 252160 Molybdenum cofactor deficiency B Metabolic

MOGS AR 601336 606056 Congenital disorder of glycosylation type IIb Metabolic

MPDU1 AR 604041 609180 Congenital disorder of glycosylation type If Metabolic

MTHFR AR 607093 236250 Homocystinuria due to MTHFR deficiency Metabolic

MTOR AD 601231 Mirzaa et al Structural

368 MTR AR 156570 250940 Homocystinuria-megaloblastic anemia, cblG complementation type Metabolic

NAGA AR 104170 609241 Schindler disease type I Metabolic

NARS2 AR 612803 616239 Combined oxidative phosphorylation deficiency 24 Metabolic

NDE1 AR 609449 614019 Lissencephaly 4 (with microcephaly) Structural

NDP X-linked 300658 310600 Norrie disease Genetic

NDUFA1 X-linked 300078 252010 Mitochondrial complex I deficiency Metabolic

NDUFA10 AR 603835 256000 Leigh syndrome Metabolic

NDUFA11 AR 612638 252010 Mitochondrial complex I deficiency Metabolic

NDUFA2 AR 602137 256000 Leigh syndrome Metabolic

NDUFAF2 AR 609653 256000 Leigh syndrome Metabolic

NDUFAF3 AR 612911 252010 Mitochondrial complex I deficiency Metabolic

NDUFAF4 AR 611776 252010 Mitochondrial complex I deficiency Metabolic

NDUFAF5 AR 612360 252010 Mitochondrial complex I deficiency Metabolic

ILAE Gene Inheritanc OMIM OMIM gene Condition name classification name^ e pattern phenotype* group

NDUFS1 AR 157655 252010 Mitochondrial complex I deficiency Metabolic

NDUFS2 AR 602985 252010 Mitochondrial complex I deficiency Metabolic

NDUFS4 AR 602694 multiple Leigh syndrome (256000), Mitochondrial complex I deficiency (252010) Metabolic phenotype IDs

NDUFS6 AR 603848 252010 Mitochondrial complex I deficiency Metabolic

NDUFS7 AR 601825 256000 Leigh syndrome Metabolic

NDUFS8 AR 602141 256000 Leigh syndrome Metabolic

NDUFV1 AR 161015 252010 Mitochondrial complex I deficiency Metabolic

NECAP1 AR 611623 615833 EIEE21 Genetic

NEDD4L AD 606384 Broix et al Periventricular nodular heterotopia Structural

369

NGLY1 AR 610661 615273 Congenital disorder of deglycosylation Metabolic

NHLRC1 AR 608072 254780 Progressive myoclonic epilepsy 2B (Lafora) Genetic

NPRL2 AD 607072 617116 Familial focal epilepsy with variable foci 2 Structural AND Genetic

NPRL3 AD 600928 617118 Familial focal epilepsy with variable foci 3 Structural AND Genetic

NRAS AD 164790 multiple Noonan syndrome 6 (613224), Neurocutaneous melanosis (249400) Structural phenotype IDs

NRXN1 AR 600565 614325 Pitt-Hopkins-like syndrome 2 Genetic

NSD1 AD 606681 117550 Sotos syndrome Genetic

NSDHL X-linked 300275 300831 CK syndrome Structural

NUBPL AR 613621 252010 Mitochondrial complex I deficiency Metabolic

OCLN AR 602876 251290 Band-like calcification with simplified gyration and polymicrogyria Structural

ILAE Gene Inheritanc OMIM OMIM gene Condition name classification name^ e pattern phenotype* group

OPHN1 X-linked 300127 300486 Mental retardation, X-linked with cerebellar hypoplasia and distinctive Structural facial appearance

OTX2 AD 600037 610125 Microphthalmia, syndromic 5 Structural

PAFAH1B1 AD 601545 607432 Lissencephaly 1 Structural

PAH AR 612349 261600 Phenylketonuria Metabolic

PAX6 AD 607108 just CNVs Structural pathogenic?

PCCA AR 232000 606054 Propionic acidemia Metabolic

PCCB AR 232050 606054 Propionic acidemia Metabolic

PCDH19 X-linked 300460 300088 Epilepsy limited to females with mental retardation Genetic (male

370 sparing

unless male mosaic)

PCLO AR 604918 608027 Pontocerebellar hypoplasia type 3 Structural

PDHA1 X-linked 300502 312170 Pyruvate dehydrogenase E1-alpha deficiency Metabolic

PDHX AR 608769 245349 Lacticacidemia due to PDX1 deficiency Metabolic

PDSS2 AR 610564 614652 Coenzyme Q10 deficiency, primary 3 Metabolic

PET100 AR 614770 220110 Mitochondrial complex IV deficiency Metabolic

PEX1 AR 602136 214100 Zellweger syndrome Metabolic

PEX7 AR 601757 215100 Rhizomelia chondrodysplasia punctata, type 1 Metabolic

PHGDH AR 606879 601815 Phosphoglycerate dehydrogenase deficiency Metabolic

PIGA X-linked 311770 300868 Multiple congenital anomalies-hypotonia-seizures syndrome 2 Genetic

PIGO AR 614730 614749 Hyperphosphatasia with mental retardation syndrome 2 Genetic

ILAE Gene Inheritanc OMIM OMIM gene Condition name classification name^ e pattern phenotype* group

PIGQ AR 605754 SINGLE Genetic PATIENT

PIGW AR 610275 616025 Hyperphosphatasia with mental retardation syndrome 3 Genetic

PIK3CA AD 171834 602501 Megalencephaly-capillary malformation-polymicrogyria syndrome Structural

PIK3R2 AD 603157 603387 Megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome 1 Structural

PLCB1 AR 607120 613722 EIEE12 Genetic

PMM2 AR 601785 212065 Congenital disorder of glycosylation type 1a Metabolic

PNKP AR 605610 613402 Microcephaly, seizures and developmental delay Structural

PNPO AR 603827 610090 Pyridoxamine 5-prime-phosphate oxidase deficiency Metabolic

POLG AR 174763 203700 Alpers syndrome Metabolic

371

POMGNT1 AR 606822 253280 Muscular dystrophy-dystroglycanopathy (congenital with brain and eye Structural anomalies), type A3

POMT1 AR 607423 236670 Muscular dystrophy-dystroglycanopathy (congenital with brain and eye Structural anomalies), type A1

POMT2 AR 607439 613150 Muscular dystrophy-dystroglycanopathy (congenital with brain and eye Structural anomalies), type A2

PPT1 AR 600722 256730 Neuronal ceroid lipofuscinosis 1 Metabolic

PRICKLE1 AR 608500 612437 Progressive myoclonic epilepsy 1B Genetic

PRRT2 AD 614386 Heron et al Benign familial infantile seizures 2 (605751), Familial infantile Genetic convulsions with paroxysmal choreoathetosis (602066)

PSAP AR 176801 multiple Combined SAP deficiency (611721), Metachromatic leukodystrophy due Metabolic phenotype IDs to SAP-b deficiency (249900)

PSAT1 AR 610936 610992 Phosphoserine aminotransferase deficiency Metabolic

PSPH AR 172480 614023 Phosphoserine phosphatase deficiency Metabolic

ILAE Gene Inheritanc OMIM OMIM gene Condition name classification name^ e pattern phenotype* group

PTEN AD 601728 158350 Cowden syndrome 1 Structural

PTF1A AR 607194 609069 Pancreatic and cerebellar agenesis Structural

PTS AR 612719 261640 Hyperphenylalaninemia, BH4-deficiency A Metabolic

PURA AD 600473 616158 Mental retardation, autosomal dominant 31 Genetic

QARS AR 603727 615760 Microcephaly, progressive, seizures and cerebral and cerebellar atrophy Genetic

QDPR AR 612676 261630 Hyperphenylalaninemia, BH4-deficiency C Metabolic

RAB18 AR 602207 614222 Warburg micro syndrome 3 Structural

RAB3GAP1 AR 602536 600118 Warburg micro syndrome 1 Structural

RAB3GAP2 AR 609275 614225 Warburg micro syndrome 2 Structural

372 RARS2 AR 611524 611523 Pontocerebellar hypoplasia type 6 Structural

RELN AR 600514 257320 Lissencephaly 2 Structural

RMND1 AR 614917 614922 Combined oxidative phosphorylation II Metabolic

RNASEH2A AR 606034 610333 Aicardi-Goutieres syndrome 4 Genetic

RNASEH2B AR 610326 610181 Aicardi-Goutieres syndrome 2 Genetic

RNASEH2C AR 610330 610329 Aicardi-Goutieres syndrome 3 Genetic

RNASET2 AR 612944 612951 Leukoencephalopathy, cystic, without megalencephaly Structural

RNU4ATAC AR 601428 210710 Microcephalic osteodysplastic primordial dwarfism, type 1 Structural

ROGDI AR 614574 226750 Kohlschutter-Tonz syndrome Genetic

RRM2B AR 604712 612075 Mitochondrial DNA depletion syndrome 8A (encephalomyopathic type Metabolic with renal tubulopathy)

SAMHD1 AR 606754 612952 Aicardi-Goutieres syndrome 5 Genetic

SCN1A AD 182389 607208 EIEE6 Genetic

SCN1B AD/?AR 600235 604233 Generalised epilepsy with febrile seizures plus type 1 Genetic

ILAE Gene Inheritanc OMIM OMIM gene Condition name classification name^ e pattern phenotype* group

SCN2A AD 182390 613721 EIEE11 Genetic

SCN8A AD 600702 614558 EIEE13 Genetic

SCO1 AR 603644 220110 Mitochondrial complex IV deficiency Metabolic

SCO2 AR 604272 604377 Cardioencephalopmyopathy, fatal infantile, due to cytochrome c oxidase Metabolic deficiency 1

SDHA AR 600857 multiple Leigh syndrome (256000), mitochondrial respiratory chain complex II Metabolic phenotype IDs deficiency

SEPSECS AR 613009 613811 Pontocerebellar hypoplasia type 2D Structural

SETBP1 AD 611060 multiple Schinzel-Giedion midface retraction syndrome (269150), mental Genetic phenotype IDs retardation, autosomal dominant 29 (616078)

SHH AD 600725 142945 Holoprosencephaly 3 Structural

373

SIK1 AD 605705 616341 EIEE30 Genetic

SIX3 AD 603714 157170 Holoprosencephaly 2 Structural

SLC13A5 AR 608305 615905 EIEE25 Genetic

SLC25A12 AR 603667 612949 EIEE39 Genetic

SLC25A22 AR 609302 609304 EIEE3 Genetic

SLC2A1 AD 138140 606777 GLUT1 deficiency syndrome Metabolic

SLC35A2 X-linked 314375 300896 Congenital disorder of glycosylation type Iim Metabolic

SLC6A1 AD 137165 616421 Myoclonic-atonic epilepsy Genetic

SLC6A8 X-linked 300036 300352 Cerebral creatine deficiency syndrome 1 Metabolic

SLC9A6 X-linked 300231 300243 Christianson syndrome Genetic

SMC1A X-linked 300040 300590 Cornelia de Lange syndrome 2 Genetic

SPR AR 182125 612716 Sepiapterin reductase deficiency Metabolic

ILAE Gene Inheritanc OMIM OMIM gene Condition name classification name^ e pattern phenotype* group

SPTAN1 AD 182810 613477 EIEE5 Genetic

ST3GAL3 AR 606494 615006 EIEE15 Metabolic

ST3GAL5 AR 604402 609056 Salt and pepper developmental regression syndrome Metabolic

STAMBP AR 606247 614261 Microcephaly-malformation syndrome Structural

STRADA AR 608626 611087 Polyhydramnios, megalencephaly and symptomatic epilepsy Structural

STXBP1 AD 602926 612164 EIEE4 Genetic

SUCLA2 AR 603921 612073 Mitochondrial DNA depletion syndrome 5 (encephalomyopathic with or Metabolic without methylmalonic aciduria)

SUCLG1 AR 611224 245400 Mitochondrial DNA depletion syndrome 9 (encephalomyopathic with or Metabolic without methylmalonic aciduria)

374

SUOX AR 606887 272300 Sulfite oxidase deficiency Metabolic

SURF1 AR 185620 256000 Leigh syndrome Metabolic

SYNGAP1 AD 603384 612621 Mental retardation, autosomal dominant 5 Genetic

SZT2 AR 615463 615476 EIEE18 Genetic

TBC1D20 AR 611663 615663 Warburg micro syndrome 4 Structural

TBC1D24 AR 613577 multiple DOOR syndrome (220500), EIEE16 (615338), Familial infantile Genetic phenotype IDs myoclonic epilepsy (605021)

TBL1XR1 AD 608628 multiple Pierpont syndrome (602342), Mental retardation, autosomal dominant 41 Genetic phenotype IDs (616944)

TCF4 AD 602272 610954 Pitt-Hopkins syndrome Genetic

TMEM70 AR 612418 614052 Mitochondrial complex V (ATP synthase) deficiency, nuclear type 2 Metabolic

TNK2 AR 606994 Hitomi et al Genetic

TPP1 AR 607998 204500 Neuronal ceroid lipofuscinosis 2 Metabolic

TREX1 AD/AR 606609 225750 Aicardi-Goutieres syndrome Genetic

ILAE Gene Inheritanc OMIM OMIM gene Condition name classification name^ e pattern phenotype* group

TSC1 AD 605284 191100 Tuberous sclerosis 1 Structural

TSC2 AD 191092 613254 Tuberous sclerosis 2 Structural

TSEN2 AR 608753 612389 Pontocerebellar hypoplasia type 2B Structural

TSEN34 AR 608754 612390 Pontocerebellar hypoplasia type 2A Structural

TSEN54 AR 608755 277470 Pontocerebellar hypoplasia type 2C Structural

TSFM AR 604723 610505 Combined oxidative phosphorylation deficiency 3 Metabolic

TUBA1A AD 602529 611603 Lissencephaly 3 Structural

TUBA8 AR 605742 613180 Polymicrogyria with optic nerve hypoplasia Structural

TUBB AD 191130 615771 Cortical dysplasia, complex, with other brain malformations Structural

375 TUBB2B AD 612850 610031 Polymicrogyria, symmetric or asymmetric Structural

TUBB3 AD 602661 614039 Cortical dysplasia, complex, with other brain malformations 1 Structural

TUBB4A AD 602662 612438 Hypomyelinating leukodystrophy 6 Structural

TUBG1 AD 191135 615412 Cortical dysplasia, complex, with other brain malformations 4 Structural

UBE3A AD 601623 105830 Angelman syndrome Genetic

VLDLR AR 192977 224050 Cerebellar ataxia and mental retardation witho or without quadrupedal Genetic locomotion 1

WDR45 X-linked 300526 300894 Static encephalopathy of childhood with neurodegeneration in adulthood Genetic

WDR62 AR 613583 604317 Microcephaly 2, primary, autosomal recessive, with or without cortical Structural malformations

WWOX AR 605131 616211 EIEE28 Genetic

ZEB2 AD 605802 235730 Mowat-Wilson syndrome Genetic ^HGNC approved name

*OR reference to paper if no OMIM phenotype OR note of single case where relevant

376

Appendix E: Variant classification scheme for whole exome sequencing

A: Pathogenic/likely pathogenic

Criteria:

 Mutation in a gene consistent with phenotype that is either: o Protein truncating variant (PTV) affecting a known functional domain (except where PTVs frequent in population datasets) OR o Missense variant that is . Previously described in literature  In individuals with same phenotype OR  With convincing segregation plus/minus  With functional data from an established assay OR

. Novel  With strong support from in silico analysis or segregation analysis plus/minus  With functional data from an established assay Action:

 Report to clinician.  Clinician to arrange testing via a NATA-approved source for confirmation.  Once confirmed, genotype-dependent management and reproductive counselling. B: Potentially pathogenic

Criteria:

 Mutation in a gene that is possibly consistent with the phenotype that is either: o PTV affecting a known functional domain (except where PTVs frequent in population datasets) OR o Missense variant  With strong support from in silico analysis or segregation analysis plus/minus

377

 With functional data from an established assay Action:

 Report to clinician for further clinical investigation to establish pathogenicity (specific patient investigations, family studies to correlate genotype with phenotype, other)  Involve geneticist to make call on implications for reproductive counselling C: Variant of unknown significance

Criteria:

 Missense variant: o In a gene consistent with phenotype that: . Is reported <1:100 in population OR . Inconsistent support from segregation studies and/or functional assays . Plus/minus has inconsistent support from in silico analysis, OR o In a gene not consistent with phenotype OR  PTV in a gene consistent with phenotype, where PTVs are frequent in population datasets or not known to affect a functional domain Action:

 Consider further efforts at classifying mutation such as identifying other patients with same mutation, or by functional studies (via research or consortia where available).  Consider re-phenotyping.  Not sufficient for reproductive counselling. D: Likely neutral

Criteria:

 Variant with consistent evidence of neutrality from databases (eg previous reports of variant in unaffected patients), in silico analysis, segregation or functional studies) Action:

 Treat as no mutation found.

378

Appendix F: Criteria for approval of a waiver of consent for the ascertainment phase of this study

The information below, which was submitted as part of the applications for ethics approval of this project, is required by the Human Research and Ethic Committees of Australian Hospitals in order to waive consent to access patient records for research purposes. A waiver is considered for low risk research where the benefits of research justify any issues of not seeking consent and where it is impractical to obtain consent.

Indication for waiver of consent for the screening phase of ‘The epidemiology and genetics of the infantile epileptic encephalopathies’ study

Addressing the criteria outlined in Section 2.3.6 of the National Statement: (a) Involvement in the research carries no more than low risk (see paragraphs 2.1.6 and 2.1.7, page 18) to participants The investigators believe that the screening phase of the study carries no risk. The screening phase does not involve trialling an intervention, invasive procedures, collection of tissue or blood products, genetic testing, research which may show unknown disabilities/disease/non-paternity or use of ionising radiation. The identifiable personal or health information that will be viewed +- collected without participant consent is akin to that collected in a clinical audit. Collection of screening phase data does not involve participant contact, hence will not inconvenience them. All identifying data that is assessed or collected will be kept confidential. (b) The benefits from the research justify any risks of harm associated with not seeking consent This study will provide epidemiologic data on the relative proportion and incidence of the emerging new genetic causes of IEE. This data is vital to understanding the contribution of each gene to IEE at a population level and to how best to apply new genetic technologies to the diagnosis of IEE. (c) It is impracticable to obtain consent (for example, due to the quantity, age or accessibility of records) It is impractical to obtain consent for this phase of the study as it involves screening the records of thousands of infants who have undergone EEGs or had seizures in neonatal units. The majority of these children will have benign clinical situations; in most of those, that will be clear in these searches. For those children, no further information collection will be required, and no identifying information will be recorded. Attempting to contact all these families would be prohibitive, and there is no need to bother them.

379

For children identified to have a possible IEE from these searches, basic identifying information will be recorded for epidemiologic purposes. Some of these will be duplicate identifications, having been identified through other screening methods also. The investigators will approach the treating doctor of children identified as having possible IEE of genetic, possible genetic or unknown cause through these searches to invite their participation in the assessment phase of the study. (d) There is no known or likely reason for thinking that participants would not have consented if they had been asked The information collected is akin to that collected in a clinical audit. As such, it is reasonable that the participant may expect that information be used and accessed in this way. (e) There is sufficient protection of their privacy The information collected will be kept confidentially and accessed only by the study investigators. No identifying information will be recorded on the patients in whom the screened records do not suggest that the patient has an IEE. The only information collected will be the total number of records searched, and basic identifying information on patients identified in these searches as having potential IEE. No identifying information will be disclosed. (f) There is an adequate plan to protect the confidentiality of data Identifying data will be kept confidentially. Paper records will be kept in a locked drawer, computer records in password-protected files on a password-protected, user- restricted departmental server. Both will only be accessible by study investigators. No identifying information will be disclosed. The information collected in the screening phase of the study will be kept for the duration of the study and for twelve months after anticipated publication of the study findings. Following this, screening data will be destroyed. The only information maintained will be on patients consented for inclusion in the assessment phase of the study. (g) In case the results have significance for the participants’ welfare there is, where practicable, a plan for making information arising from the research available to them (for example, via a disease-specific website or regional news media) This is not relevant to the screening phase of the study. It is relevant to the assessment phase of the study, and this is covered by RCH ethics approval. (h) The possibility of commercial exploitation of derivatives of the data or tissue will not deprive the participants of any financial benefits to which they would be entitled Commercial exploitation of derivatives of data or tissue is not anticipated. (i) The waiver is not prohibited by State, federal, or international law. The investigators do not believe that a waiver in this instance violates any State, federal or international law.

380 Appendix G: Human Research and Ethics Committee approval letters

This appendix contains approval letters from the Human Research and Ethics Committees of the Royal Children’s Hospital, Monash Health, Austin Hospital, Royal Women’s Hospital, Mercy Hospital for Women and Geelong Hospital.

381

RCH HUMAN RESEARCH ETHICS COMMITTEE APPROVAL

HREC REF. No: 32288 C PROJECT TITLE: Severe infantile epilepsy study/ IEE study

DOCUMENTS APPROVED: Permission to Contact Letter v1 dated 31 January 2014

APPROVED PROTOCOL: Protocol v3.2 dated 31 January 2014

PRINCIPAL INVESTIGATOR: Katherine Howell

DATE OF MODIFICATION APPROVAL: 13 February 2014 DURATION: 37 months DATE OF APPROVAL EXPIRY: 8 March 2017

SIGNED: ...... 13th February 2014 COMMITTEE REPRESENTATIVE

APPROVED SUBJECT TO THE FOLLOWING CONDITIONS: ALL PROJECTS 1. The study must not commence until all Research Agreements have been executed (if applicable) 2. Must comply with the Investigator’s Responsibilities in Research Procedure and other Campus Research Policies and Procedures 3. Any proposed change in the protocol or approved documents or the addition of documents must be submitted to the Human Research Ethics Committee (HREC) for approval prior to implementation, including: - flyers, brochures, advertising material - Increase in recruitment target 4. The Principal Investigator must notify Research Development & Ethics of: - Any serious adverse effects of the study on participants and steps taken to deal with them. - Any unforeseen events (e.g. protocol violations or complaints). - Investigators withdrawing from or joining the project. 5. A progress report must be submitted annually and at the conclusion of the project. 6. RCH HREC approval must remain current for the entire duration of the project. If the project is not completed in the allocated time a renewal request must be submitted to the Research Development & Ethics. Investigators undertaking projects without current HREC approval risk their indemnity, funding and publication rights. CLINICAL TRIALS 7. Must comply with Good Clinical Practice (GCP) 8. Must report all internal (occurring in RCH participants) Serious Adverse Events (SAE) to the sponsor and the RCH HREC within 72 hours of occurrence. 9. Must report all Suspected Unexpected Serious Adverse Reactions (SUSARS) to the Therapeutic Goods Administration (TGA) (for sponsored studies the sponsor may take this responsibility).

Date: 5 June 2013 To: Dr Katherine Howell Neurology Department Royal Children's Hospital Project: The incidence & genetics of infantile epileptic encephalopathies Project No: H2013/05082 RCH HREC Ref No: 32288 Approval period 5 June 2013 to 5 June 2016

Thank you for submitting a project for consideration under Mutual Acceptance for authorisation at Austin Health. I can confirm that the submission was received on 5 June 2013.

I am pleased to inform you that authorisation has been granted for this project to be conducted at Austin Health. The period of authorisation for this study to be conducted at Austin Health is 3 years from the date of this letter. Authorisation for this study will expire on the 05 June 2016. If you wish to continue with this study at this site past this date, a request for extension should be submitted to Austin Health Research Ethics. This extension request should provide justification as to why the study should continue at this site.

We have been advised that the following other sites will be participating in this study:

Royal Children’s Hospital Melbourne Monash Medical Centre Royal Women’s Hospital Mercy Hospital for Women Victorian EEG laboratories (Bendigo, Ballarat, Geelong, Shepparton, Warrugal & Frankston) Austin Health

The following conditions apply to this research project at Austin Health. These conditions are additional to those imposed by the Human Research Ethics Committee that granted ethical approval.

Before the study can commence you must ensure that you have: In addition to the reporting requirements of the reviewing HREC, you are required to submit an Austin Health specific yearly progress report and an Austin Health specific final report at the completion of the study. The annual reporting period will be in line with the financial year, the period July 1 – June 30, with submission to the Office for Research due by the end of September each year. Research Ethics will accept annual progress reports with the coversheet on the template provided at: www.health.vic.gov.au/clinicaltrials If your study will not commence within 12 months, please notify the Austin Health Office For Research in addition to the reviewing HREC. After commencement of your study, should the trial be discontinued prematurely you must notify Austin Health Office for Research of this, citing the reason. Any changes to the original application will require a submission of a protocol amendment to the reviewing HREC in addition to site-specific approval. Authorisation of this project only relates to the original application as detailed above. Please notify the Austin Health Office for Research of any changes to research personnel. All new investigators must be approved prior to performing any study related activities. It is now your responsibility to ensure that all people (i.e. all investigators, sponsor and other relevant departments in the hospital) associated with this particular study are made aware of what has been approved.

Page 1 of 2 V1 250313 SH Please inform the Austin Health Office for Research ([email protected]) of the actual starting date of the study as soon as the study commences. A written notice (e-mail, fax or letter) is considered the appropriate format for notification. Please ensure you frequently refer to the Research Ethics website http://www.austin.org.au/researchethics/ for all up to date information about research and governance requirements.

Document Version Date NEAF 16 May 2013 IEE study protocol 3.1 22 April 2013 Study diagram 22 April 2013 Victorian Specific Module 15 May 2013 PGIS & CF 4 March 2013 PGIS & CF (involvement of parent after participant has died) 7 7 March 2013 Cover Letter 18 Jan 2013 Cover Letter (for deceased child) 3 7 March 2013 Expression of interest form 18 Jan 2013 Letter to paediatricians 11 Feb 2013 Reminder Letter Treating doctor permission form Austin Health Site Specific Assessment Form 1 25 May 2013 Health Information Services Declaration 30 May 2013

If you have any matters that arise regarding conduct of the research at this site, please ensure you contact the Research Governance Officer.

Yours sincerely,

Dr Sianna Panagiotopoulos, PhD Manager, Office for Research

This HREC is constituted and operates in accordance with the National Health and Medical Research Council’s (NHMRC) National Statement on Ethical Conduct in Human Research (2007), NHMRC and Universities Australia Australian Code for the Responsible Conduct of Research (2007) and the CPMP/ICH Note for Guidance on Good Clinical Practice annotated with TGA comments (July 2008) and the applicable laws and regulations; and the Health Privacy Principles in The Health Record Act 2001. The process this HREC uses to review multi-centre research proposals has been certified by the NHMRC.

Page 2 of 2 V1 050613 SH Mr Arthur Hui Administrative Officer Research and Ethics Secretariat Tel: +61 3 8345 3720 Fax: +61 3 8345 3702 Email: [email protected]

27.8.13

Dr K Howell Department of Neurology RCH

Dear Dr Howell,

Re: Project 13/24 - The incidence and genetics of the infantile epileptic encephalopathies

Thank you for submitting the clarification and amendments as requested by the RWH Human Research Ethics Committee.

I confirm the project is now approved.

Enclosed please find Project Approval and Notification of Project Commencement Forms for your record.

Prior to commencement of your project, you are reminded that you must contact the relevant RWH Divisional Directors / Department Heads to confirm your actual commencement date. Failure to inform these RWH personnel may jeopardise their approval and support for your project.

Please return the completed Notification of Project Commencement Form to me when the project begins.

Yours sincerely,

A. C. B. Hui Administrative Officer Research and Ethics Secretariat

Encl:

cc A/Prof S Jacobs

THE ROYAL WOMEN’S HOSPITAL

RESEARCH AND HUMAN RESEARCH ETHICS COMMITTEES

PROJECT APPROVAL

PROJECT NO: 13/24

PROJECT TITLE: The incidence and genetics of the infantile epileptic encephalopathies

INVESTIGATOR (S): K Howell, S Harvey, I Scheffer, S Jacobs, M Hayman, F Wong, J Holberton

DATE OF APPROVAL: 27 August 2013

DURATION: Thirty (30) months

SIGNED …………………………………………………………………. Secretary, Research & Human Research Ethics Committees DATE

CONDITIONS OF APPROVAL

The Principal Investigator is reminded of the following:-

1. Prior to commencement of the project, you must contact the relevant RWH Divisional Directors / Department Heads to confirm your actual commencement date. Failure to inform these RWH personnel may jeopardise their approval and support for your project.

2. A Project may commence once the Principal Investigator has received written confirmation that the Human Research Ethics Committee has approved the Project.

3. Substantial changes in protocols must be submitted to the Research/Human Research Ethics Committees for approval.

4. Progress reports must be submitted annually. A request will be forwarded to the Principal Investigator. If no report is supplied, permission to continue the project may lapse.

5. The Research/Human Research Ethics Committees must be notified IMMEDIATELY of any untoward or unexpected complications or side affects arising during the project or of any ethical or medico-legal problems that may arise.

6. Consent forms must be available for audit and retained on file for five (5) years.

7. Raw data and details of analysis must be retained by the Principal Investigator for five (5) years.

8. Principal Investigator MUST upon leaving the Institution, inform the Human Research Ethics Committee as to the nominated person to replace him/her.

PLEASE QUOTE PROJECT NO. AND TITLE FOR ALL CORRESPONDENCE

RWH PROJECT NUMBER 13/24

THE ROYAL WOMEN’S HOSPITAL

RESEARCH AND HUMAN RESEARCH ETHICS COMMITTEES

NOTIFICATION OF PROJECT COMMENCEMENT

PROJECT TITLE: The incidence and genetics of the infantile epileptic encephalopathies

INVESTIGATOR (S): K Howell, S Harvey, I Scheffer, S Jacobs, M Hayman, F Wong, J Holberton

DATE OF APPROVAL: 27 August 2013

DURATION: Thirty (30) months

DATE OF COMMENCEMENT ……...... /.……...... /…......

PRINCIPAL INVESTIGATOR:

NAME...... ( PLEASE PRINT )

SIGNATURE...... DATE……...... /..……..../......

13 March 2015

Barwon Health Reference 15/17 Project Title A statewide population-based study of severe epilepsy in infancy Principal Researcher Katherine Howell Research Team Dr A Simon Harvey, Prof Ingrid Scheffer, Mr Mark Mackay

The Barwon Health Research Ethics, Governance & Integrity (REGI) Unit advises that the above project is exempt from ethical review as outlined in the National Statement on Ethical Conduct in Human Research Section 5. Conduct of the project is subject to compliance with the conditions set out and any additional conditions specified by the Barwon Health Research Governance Committee.

The documents reviewed and approved are:

Acknowledgement of Prior HREC Approval NEAF – Submission Code dated 16/05/2013 SSA – Submission Code dated 09/02/2015 Protocol Version 3.2 dated 31/01/2014 Study Design Diagram Victorian Specific Module

In order to comply with the National Statement on Ethical Conduct in Human Research (2007), Guidelines for Good Clinical Research Practice and local research policies and guidelines, you are required to notify the Research Ethics, Governance & Integrity (REGI) Unit of: . The actual start date of the project at Barwon Health; . Any amendments to the project after these have been approved by the reviewing HREC; . Any adverse events or unexpected developments in the project with ethical implications; . Your inability to continue as Principal Investigator and any other change in research personnel involved in the project at Barwon Health; . Any decision taken to end the project prior/after the expected date of completion, and; . To provide a comprehensive written annual/final report/s on the anniversary of project approval advising of the progress of the project and a final report advising of completion.

Templates for reporting changes to the project and annual/final reports may be obtained from the Department of Health website - http://www.health.vic.gov.au/clinicaltrials/application-instructions.htm

Special Conditions The site/s to which this approval pertains includes:

Barwon Health

Additional Conditions Barwon Health reserves the right to conduct an audit of this project at any time.

Please quote the Barwon Health reference number and title in all future correspondence.

The approval of this project will be ratified at the upcoming Human Research Ethics Committee (HREC) meeting. On behalf of the Barwon Health Research Ethics, Governance & Integrity (REGI) Unit, best wishes for your research project.

Yours sincerely

Dr Giuliana Fuscaldo Chair – Research Review Committee Manager – Research Ethics, Governance & Integrity (REGI) Unit Barwon Health

All correspondence to: Research Ethics, Governance & Integrity (REGI) Unit, Barwon Health – Post. PO Box 281 Geelong 3220 Phone: (03) 4215 3372 Email: [email protected]

Appendix H: Parent information and consent forms

Two versions of the parent information and consent form are included here. The first was given to parents of living infants and the second to parents of deceased infants.

395

The Royal Children’s Hospital, Melbourne

Title The incidence and genetics of the infantile epileptic encephalopathies Short Title Severe epilepsy in infants Protocol Number Version 4.0 Coordinating Principal Investigator/ Dr Katherine Howell Principal Investigator Associate Investigator(s) Dr Simon Harvey, Prof Ingrid Scheffer

Location The Royal Children’s Hospital, Melbourne

1 Introduction

This is an invitation for your child to take part in this research project. Your child has (or has had) seizures or an abnormal EEG, and may be eligible for inclusion in the research. The research project aims to understand the occurrence and causes of epilepsies in infants.

This Participant Information Sheet/Consent Form tells you about the research project. It explains the tests and research involved. Knowing what is involved will help you decide if you want your child to take part in the research.

Please read this information carefully. Ask questions about anything that you don’t understand or want to know more about. Before deciding whether or not your child can take part, you might want to talk about it with a relative, friend or local doctor.

Participation in this research is voluntary. If you do not wish for your child to take part, they do not have to. They will receive the best possible care whether or not they take part.

If you decide you want your child to take part in the research project, you will be asked to sign the consent section. By signing it you are telling us that you: • Understand what you have read • Consent to your child taking part in the research project • Consent to your child having the tests and research that are described • Consent to the use of your child’s personal and health information as described. You will be given a copy of this Participant Information and Consent Form to keep.

2 What is the purpose of this research?

Some of the epilepsies (seizure disorders) that affect children in the first 18 months of life are known as ‘infantile epileptic encephalopathies’ (IEE). These are typically characterised by frequent seizures, an abnormal electroencephalogram (EEG) and concerns about the infant’s development. The cause of IEE is unknown in many infants, but presumed to be genetic.

As a first step in this project, we did a review of RCH patient medical records. The Health Records Act lets us collect and examine clinical information from hospital medical records to monitor and improve services. We are able to collect information to help us learn more about the causes and how often IEE happens in children. This information has been recorded in a research database and is used in a way that does not identify your child

This study of children in Victoria aims to determine the number of children diagnosed with IEE each year, understand the various causes of IEE and determine characteristic features of each of the different IEE. Understanding the causes of IEE, particularly genetic causes, is a key step towards development of potential treatments.

The results of this research will be used by the principal investigator, Dr Katherine Howell, to obtain a Doctor of Philosophy higher degree. This research is being conducted by The Department of Neurology at The Royal Children’s Hospital, Melbourne and the Epilepsy Research Centre based at Austin Health, and The Department of Paediatrics, University of Melbourne.

3 What does participation in this research involve?

Medical assessment The assessment will involve an interview to collect details of your child’s seizures, developmental and other medical history. There will be a physical examination, which will involve checking your child’s reflexes and strength and examining their eyes. A standardised developmental assessment involving an interview about your child’s current abilities will also be performed. Each assessment will be performed at The Royal Children’s Hospital and take approximately two hours in total. Children in whom a diagnosis of IEE is confirmed will be reviewed twice a year while your child is under two years old and once a year thereafter to understand how their condition changes over time if you are happy for this to occur.

Routine clinical investigations Your child may have undergone many investigations to identify the cause of their epilepsy. These may have been performed at institutions other than The Royal Children’s Hospital. The study investigators will review any investigations your child has already had at other institutions, including their EEG recordings, MRI scans and any testing of blood, urine and cerebrospinal fluid. Where standard clinically-indicated investigations for infants with epilepsy have not been performed, we will suggest to you and your child’s treating doctor that these be undertaken. Suggested tests are not compulsory. Your child’s treating doctor will decide, in discussion with you, whether these will be performed. These tests are looking for known causes of IEE, including some important treatable conditions.

The assessments and any testing can be scheduled at convenient times for you and your child, and broken up if necessary. For families living in rural areas, some of the assessment can be performed with telehealth technology.

Optional consent:

Blood tests for genetic testing If the cause of your child’s epilepsy is unknown, a blood sample will be requested to look for genes that cause epilepsy in infants and young children. In some cases, your child may have already provided a blood or tissue sample for genetic testing. If this sample is available, we require your consent to use it in this project. If possible, a blood sample will also be requested from each parent, in order to determine the significance of any gene change found in your child. Up to 40ml (2 tablespoons) of blood will be collected from adults, and up to 5ml (1 teaspoon) in young infants. All the blood required for this study will usually be taken at once, so no additional blood tests are

Parent information statement and consent form Version 4.0, March 2013 Page 2 of 10 needed. A saliva or cheek brushing sample may be given if a blood sample is not able to be collected. The samples, identified with their name, date of birth and clinical diagnosis, will be sent to our laboratory at the Epilepsy Research Centre, University of Melbourne, and DNA (genetic material) will be removed from the blood for testing. Genetic testing for known and new epilepsy genes will often be performed in interstate or overseas research laboratories that collaborate with our team. We ask you to also consider:  allowing us to use the blood we collect from your child to grow a culture of cells. This will provide an ongoing supply of their DNA (genetic material) so we can do more testing than we would be able to from a normal blood sample, as new genes are discovered.  allowing us to store and use DNA samples for future ethically approved research studies related to epilepsy. You can choose whether you would like to be informed when your child’s sample is used again.  allowing us to include your child’s data and DNA samples in a larger, related study, ‘Genetic Basis of Epilepsy’ (RCH HREC 20114), that is also looking at genes that cause all forms of epilepsy.

There are no costs associated with participating in this research project, nor will you or the participant be paid. Reasonable parking and travel costs will be reimbursed. You do not stand to gain financially from participation even if useful scientific discoveries are made.

Your consent for your child’s participation in this study will be obtained prior to any study assessments being performed.

4 What does my child have to do?

Participants will be required to attend assessments and, if required and agreed to, have a blood test. This study places no restrictions or conditions on lifestyle, diet or medications.

5 Other relevant information about the research project

This research project is a Victoria-wide study. Up to 500 infants may be identified as potentially eligible for this study during its period of recruitment, and up to 300 are expected to ultimately meet criteria for inclusion in the study.

6 Does my child have to take part in this research project?

Participation in any research project is voluntary. If you do not wish for your child to take part, they do not have to. If you decide that they can take part and later change your mind, you are free to withdraw them from the project at any stage.

If you do decide that your child can take part, you will be given this Participant Information and Consent Form to sign and you will be given a copy to keep.

Your decision whether your child can or cannot take part, or take part and then be withdrawn, will not affect their routine treatment, relationship with those treating them or relationship with The Royal Children’s Hospital, Melbourne.

7 What are the alternatives to participation?

Your child does not have to take part in this research project to receive treatment at this hospital.

8 What are the possible benefits of taking part?

Parent information statement and consent form Version 4.0, March 2013 Page 3 of 10 We cannot guarantee that your child will benefit from this research. However, possible benefits include identification of the cause of IEE in your child. Additionally, the knowledge gained from your child’s participation in this study may result in improvements in the treatment of other children with IEE.

9 What are the possible risks and disadvantages of taking part?

Medical assessment No risks or disadvantages other than time and inconvenience are anticipated.

Routine clinical investigations These investigations are considered standard in children with severe epilepsies. As such, while each investigation has potential risks, the risks are considered to be that of routine clinical investigation and care.

Blood tests There are no major risks associated with a blood test. The blood test may cause discomfort, bruising, minor infection or bleeding or fainting. If this happens, it can be easily treated. The collection of blood samples will not cost you money.

Genetic tests

The genetic tests we are doing are for research purposes. We will look at your genes for features relevant to the research project.

We are only searching for genes that are related to (condition), but it is possible that we may find genes responsible for other genetic conditions that you do not know about. If we find that you have any genetic condition that you do not know about, we will contact you to discuss the findings and refer you to a genetic counsellor.

On rare occasions, we may find a genetic change unrelated to the research that could have implications for your health. If changes are found in your genes, we will tell your doctor who will discuss this with you. If we do find an unusual gene change and tell you about it, your health can be managed in the most appropriate way. Please take time to consider the advantages and disadvantages of discovery of a health risk before deciding to take part in this research project.

If something is found in the genetic testing, you may need to tell your child about this at some time in the future. You will be able to discuss these issues individually with your child’s doctor.

The genetic tests we perform may tell us something about you or your wider family. This may have an impact on how family members relate to one another.

Some people in your family might want to know about your results and whether the result has implications for them. Your results will only be provided with your permission.

You may be required to inform insurance companies or employers in the future of any genetic information that you learn about yourself through this project.

By chance, we may discover that parents and children or siblings may not be biologically related. Information regarding paternity or maternity will not be available through this project.

Some people may find it distressing to receive information about their genetic make-up and future health. There is also the possibility that genetic information may be important in understanding the risks of having a child with (condition) in the future.

If you wish to discuss any personal issues or require supportive counselling as a result of your participation in this genetic study, we can refer you to an independent genetic counsellor who is available to you free of charge. We are also available to discuss any concerns you may have.

Parent information statement and consent form Version 4.0, March 2013 Page 4 of 10 To make it as easy as possible to take part in this study, the blood sample can be taken through your local doctor or pathology service if this is easier.

10 What if my child is withdrawn from this research project?

If you decide to withdraw your child from this research project, please notify a member of the research team. Your decision to withdraw will have no impact on your child’s care given by your child’s treating doctor, nor will it impact on care at The Royal Children’s Hospital Melbourne. If you withdraw during the research project, we will not collect additional personal information. However, personal information already collected will be retained to ensure that the results of the research project can be measured properly.

11 Could this research project be stopped unexpectedly?

This research project may be stopped unexpectedly for a variety of reasons, although this is very unlikely.

12 How will I be informed of the results of this research project?

If we find a change in your child’s DNA that is relevant to epilepsy, we will write to you and your child’s treating doctor to let you know we have some results. You have the option to contact us for further information. If you choose to receive your child’s results, we will explain what the results mean and give you the opportunity to ask any questions. It is important to remember that we may not always know what the results mean. If you would like to discuss the results further with someone separate from the study we can arrange for you to see an independent genetic counsellor.

Any changes found in your child’s DNA sequence may not be the only cause of their seizures or related disorder and we may not be able to predict accurately if the epilepsy could be passed on to other generations of your family. It may take years of research before we can be clear about the nature of the DNA change and its effects. If you would like to have results from the research passed on to others, for example your child’s local doctor, this can be arranged. We will not pass personal information between family members.

If your child has an unusual test result that is likely to have health implications for your child or your family, we can arrange for you to see a neurologist associated with this study or we can refer you to see a specialist in your local area who can discuss the results with you.

At the completion of the study, participants will be provided with a summary of the findings of the study. The summary will not identify individual participants.

13 What will happen to information about my child?

Information about your child’s epilepsy, developmental and other medical history may be obtained from their health records held at other health services for the purpose of this research. By signing the consent form you agree to the research team accessing health records at other institutions if they are relevant to participation in this research project.

Information about your child’s participation in this research project may be recorded in their health records.

Any information obtained for the purpose of this research project that can identify the participant will be treated as confidential and securely stored in the Neurology Department at The Royal Children’s Hospital or the Epilepsy Research Centre at the University of Melbourne until your child is at least 25 years old.

Parent information statement and consent form Version 4.0, March 2013 Page 5 of 10

Members of the research team and collaborating research groups involved with this project and The Royal Children’s Hospital Human Research Ethics Committee may access the information collected as part of this research project. Information obtained in this study will not be given to anyone outside the research team and collaborating research groups without your permission. It will be disclosed only with your permission, or as required by law.

In accordance with relevant Australian and/or Victorian privacy and other relevant laws, you have the right to request access to the information collected and stored by the research team about the participant. You also have the right to request that any information with which you disagree be corrected. Please contact the research team member named at the end of this document if you would like to access your child’s information.

It is anticipated that the results of this research project will be published and/or presented in a variety of forums. In any publication and/or presentation, information will be provided without any identifying details such as names or dates of birth. However, when our research findings are published in medical and scientific journals we may include family trees and detailed medical information. Such information may make it possible for someone who knows your child well, such as a family member, to identify your child and their medical information. This is unlikely but the possibility needs to be acknowledged. Other identifying information, such as photographs, will not be published without your specific written consent.

14 What will happen to my child’s test samples?

The collection of blood/DNA samples for genetic testing is an optional component of the research.

The blood samples used to obtain DNA for genetic testing are taken for research purposes. If you agree to the potential future use of DNA samples for ethically-approved research into epilepsy, we would like to store DNA samples for as long as possible so that we can retest the sample when new genes for epilepsy are identified in the future.

When your child turns 18 years, we will seek their consent (or yours if your child is unable to provide their own consent) to continue to store their sample. You can request that your child’s DNA sample be destroyed at any time by contacting us. The DNA sample will be stored in a coded form with the key to the code kept on a password-protected database that is only accessible to members of the research team.

The DNA samples will be stored by:

Professors Sam Berkovic and Ingrid Scheffer Epilepsy Research Centre University of Melbourne Austin Health Heidelberg VIC 3084 Telephone: (03) 9035 7330 Fax: (03) 9035 7307

We work closely with other research groups within Australia or internationally who are also trying to identify genes that cause seizures or related disorders, or who are trying to understand how the genetic changes cause seizures. This means we may send some of your child’s DNA sample to another research group. We would routinely send your child’s name with the DNA sample to ensure sample identification so that we can tell you if there are any important results. We may also send details about the type of seizures they have had and other relevant medical history or test results to assist interpretation of further testing. All collaborators are strictly bound by confidentiality requirements.

15 Complaints and compensation.

Parent information statement and consent form Version 4.0, March 2013 Page 6 of 10 If the participant suffers any injuries or complications as a result of this research project, you should contact the study team as soon as possible and you will be assisted with arranging appropriate medical treatment. If the participant is eligible for Medicare, they can receive any medical treatment required to treat the injury or complication, free of charge, as a public patient in any Australian public hospital.

If you have any concerns about the conduct of this study, you may contact the complaints contact person listed below.

16 Who is organising and funding the research?

This research project is being conducted jointly by the Department of Neurology at The Royal Children’s Hospital Melbourne, the Epilepsy Research Centre at the Austin Hospital, and the Department of Paediatrics at The University of Melbourne.

Dr Howell is supported by a scholarship from the National Health and Medical Research Council. The Epilepsy Research Centre holds several national and international research grants. No member of the research team will receive a personal financial benefit from your child’s involvement in this research project, other than their ordinary salary.

17 Who has reviewed the research project? All research in Australia involving humans is reviewed by an independent group of people called a Human Research Ethics Committee (HREC). The ethical aspects of this research project have been approved by the HREC of The Royal Children’s Hospital, Melbourne.

This project will be carried out according to the National Statement on Ethical Conduct in Human Research (2007). This statement has been developed to protect the interests of people who agree to participate in human research studies.

18 Further information and who to contact

The person you may need to contact will depend on the nature of your query. If you want any further information concerning this project or if the participant has any medical problems which may be related to involvement in the project (for example, any side effects), you can contact:

Clinical contact person Name Dr Katherine Howell Position Principal investigator Telephone 03 9345 5661 Email [email protected]

If you have any complaints about any aspect of the project, the way it is being conducted or any questions about being a research participant in general, then you may contact:

Complaints contact person Name Director Department Research Development and Ethics, The Royal Children’s Hospital Melbourne Telephone 03 9345 5044 Email [email protected]

Parent information statement and consent form Version 4.0, March 2013 Page 7 of 10

Consent Form – Parent/Guardian

The incidence and genetics of the infantile Title epileptic encephalopathies Short Title Severe infantile epilepsy study Protocol Number Version 4.0 Coordinating Principal Investigator/ Dr Katherine Howell Principal Investigator Associate Investigator(s) Dr Simon Harvey, Prof Ingrid Scheffer

Location The Royal Children’s Hospital, Melbourne

Declaration by Parent/Guardian

 I voluntarily consent for my child to take part in this research project.

 I believe I understand the purpose, extent and possible risks of my child’s involvement in this project.

 I have had an opportunity to ask questions and I am satisfied with the answers I have received.

 I understand that this project has been approved by The Royal Children’s Hospital Human Research Ethics Committee and will be carried out in line with the National Statement on Ethical Conduct in Human Research (2007).

 I understand I will receive a copy of this Parent/Guardian Information Statement and Consent form.

 I give permission for my child’s doctors, other health professionals, hospitals or laboratories outside this hospital to release information to The Royal Children’s Hospital, Melbourne concerning my child’s condition and treatment for the purposes of this project. I understand that such information will remain confidential.

Optional Consent

If eligible for genetic testing: I do I do not N/A consent to a blood/DNA sample being taken from my child for genetic testing to investigate the cause of their condition.

If blood/DNA sample collected: I do I do not N/A consent to my child’s information and blood/DNA sample being shared with the related study, ‘Genetic Basis of Epilepsy’ (RCH HREC 20114). I do I do not N/A consent to the storage of my child’s information and blood/DNA sample for use in future ethically approved research related to epilepsy, structural brain abnormalities or speech and language disorders. I do I do not N/A wish to be contacted when the blood/DNA sample is used in future research. I do I do not N/A consent to some of my child’s blood sample being used to grow a cell culture, providing an ongoing supply of their DNA.

Parent information statement and consent form Version 4.0, March 2013 Page 8 of 10

Name of Child (please print)

Name of Parent/Guardian (please print)

Signature of Parent/Guardian Date

Name of Witness* to Parent/Guardian’s Signature (please print)

Signature Date

* Witness is not to be the investigator, a member of the study team or their delegate. In the event that an interpreter is used, the interpreter may not act as a witness to the consent process. Witness must be 18 years or older.

Declaration by Study Doctor/Senior Researcher‡

I have given a verbal explanation of the research project, its procedures and risks and I believe that the parent/guardian of the participant has understood that explanation.

Name of Study Doctor/ Senior Researcher‡ (please print)

Signature Date

‡ A senior member of the research team must provide the explanation of, and information concerning, the research project.

Note: All parties signing the consent section must date their own signature

Parent information statement and consent form Version 4.0, March 2013 Page 9 of 10

Form for Withdrawal of Participation – Parent/Guardian

Location The Royal Children’s Hospital, Melbourne

Declaration by Parent/Guardian

I wish to withdraw my child from participation in the above research project and understand that such withdrawal will not affect their routine treatment, relationship with those treating them or relationship with The Royal Children’s Hospital, Melbourne.

Name of Child (please print)

Name of Parent/Guardian (please print)

Signature of Parent/Guardian Date

Declaration by Study Doctor/Senior Researcher†

I have given a verbal explanation of the implications of withdrawal from the research project and I believe that the parent/guardian of the participant has understood that explanation.

Name of Study Doctor/ Senior Researcher† (please print)

Signature Date

† A senior member of the research team must provide the explanation of, and information concerning, withdrawal from the research project.

Note: All parties signing the consent section must date their own signature.

Parent information statement and consent form Version 4.0, March 2013 Page 10 of 10

HREC Project Number: 32288

Research Project Title: Severe epilepsy in infants

Principal Researcher: Dr Katherine Howell, Neurology department

Version Number: 7 Version Date: 7 March 2013

We would like to offer our deepest sympathy for your loss and sincerely thank you for reading this information statement and considering taking part in our research project.

Infantile epileptic encephalopathies (IEE) include a range of epilepsy disorders that can affect children in the first 18 months of life. The cause of IEE is often unknown, but it is thought it might be genetic.

In this research study, we hope to find out the number of children diagnosed with IEE each year, learn more about the various causes of IEE, including if particular genes may be responsible. We hope understanding more about IEE will be a key step towards developing possible treatments. We hope up to 300 Victorian children and their parents will take part in this project.

This project is being conducted by the Neurology department at The Royal Children’s Hospital (RCH), the Epilepsy Research Centre and the Department of Paediatrics at University of Melbourne. It has been funded by a postgraduate scholarship from the National Health and Medical Research Council, and funds from The Royal Children’s Hospital Neurology Department and the Epilepsy Research Centre.

We would now like to invite you to consider taking part in this project. This would involve either coming to an interview at The Royal Children’s Hospital or if you prefer, we can do this by telephone or Skype. It will take about 2 hours and can be arranged for a time that best suits you. In the interview we will ask you for more information about your child’s seizures, growth and development, as well as details of their medical history.

As part of this research, we would like to collect and use information about any tests or procedures, including EEG studies, MRI scan and testing of blood, urine and cerebrospinal fluid, your child may have had at institutions other than The Royal Children’s Hospital. We will ask you if your child had tests completed at any other hospital or with a GP, and write to them with a copy of your consent form so they can send us a copy of any test results.

Optional consent:

There are some optional parts to this project that we would like you to consider: 1. If the cause of your child’s epilepsy was unknown and stored DNA (genetic material) sample is available, we would like to use this sample to look for genes that cause epilepsy 2. If you allow us to use your child’s DNA sample, we may also ask both parents to give us a blood sample or cheek swab sample to collect your DNA. This would be done so we can compare your DNA with your child’s, in order to understand the meaning of any change we may find in your child’s DNA. Blood samples can be collected at RCH or at a local pathology service if this is more convenient. A cheek swab kit could be sent to your home with instructions on how to collect the sample and return it to us. 3. Allowing us to continue to store your and your child’s DNA samples for use in future ethically approved research studies related to epilepsy. You can choose whether you would like to be informed when your child’s sample is used again. 4. Allowing us to include your child’s health information and DNA sample in a larger, related study called ‘Genetic Basis of Epilepsy’ (RCH HREC 20114), that is also looking at genes that cause epilepsy.

We do not expect any direct benefit to you if you take part in this project; however we hope the knowledge gained from your participation and inclusion of your child’s information may result in improvements in the treatment of other children with IEE.

We understand that discussing details of your child’s medical condition may be distressing for you. We can stop the interview at any time if you feel upset and we can refer you for supportive counselling if you need it.

The genetic tests we are doing are for research purposes. We will look at genes for features relevant to the research project. If we find a genetic change in your DNA samples that is related to epilepsy, we will write to let you know we have some results. It is your choice to contact us if you would like more information. If you choose to know the results, we will explain what they mean and give you the opportunity to ask any questions. It is important to remember that we may not always know what the results mean.

We are only searching for genes that are related to epilepsy. However, on rare occasions, we may find a genetic change unrelated to the research that could have implications for your health. If changes are found in your genes, we will contact you and refer you to the most appropriate health professional to talk about what the results mean. If we do find an unusual gene change and tell you about it, your health can be managed in the most appropriate way. Please take time to consider the advantages and disadvantages of discovery of a health risk before deciding to take part in this research project.

Because we are collecting samples of parents and children, by chance, we may discover that parents and children may not be biologically related. Information regarding paternity or maternity will not be available through this project.

If you wish to discuss any personal issues as a result of your participation in this genetic study, we can refer you to an independent genetic counsellor who is available to you free of charge. We are also available to discuss any concerns you may have.

Any information we collect for this research project that can identify you and your child will be treated as confidential. We can disclose the information only with your permission, except as required by law.

All information will be stored securely in the Neurology department at The Royal Children’s Hospital or the Epilepsy Research Centre at the University of Melbourne.

The following people may access information collected as part of this research project:  the research team involved with this project  the RCH Human Research Ethics Committee

In accordance with relevant Australian and/or Victorian privacy and other relevant laws, you have the right to access and correct the information we collect and store about you. Please contact us if you would like to access the information. Information collected for this project will be kept for at least 25 years.

If you give your consent to the storage of your child’s DNA sample and health information for use in future ethically approved research projects related to epilepsy, we plan to store the DNA samples for as long as possible so that we can retest the sample when new genes for epilepsy are identified in the future. DNA

Version: 7. Date: 07/03/2013 Page 2 of 5 (RDE 07/12: Participant) samples will be stored in a coded form with the key to the code kept on a password-protected database that is only accessible to members of the research team. You can request that your child’s DNA sample be destroyed at any time by contacting us.

All DNA samples will be stored by: Professors Sam Berkovic and Ingrid Scheffer Epilepsy Research Centre University of Melbourne Austin Health Heidelberg VIC 3084 Telephone: (03) 9035 7330

We work closely with other research groups within Australia and overseas who are also trying to identify genes or how genetic changes cause seizures. This means we may send some of your child’s DNA sample and details about their medical history to another research group. Any DNA sample that is sent will be labeled with your child’s name to ensure sample identification so that we can tell you if there are any important results. All collaborators are strictly bound by confidentiality requirements.

When we write or talk about the results of this project, information will be provided in such a way that your family cannot be identified. However, when our research findings are published in medical and scientific journals we may include family trees and detailed medical information. Such information may make it possible for someone, who knows your family well, to identify you. This is unlikely but we need to let you know it might be possible. The results will also be used by Dr Katherine Howell to obtain a Doctor of Philosophy higher degree.

At the end of the project, we will send you a summary of the overall project results.

Participation in this project is voluntary. You do not have to take part if you do not want to. You can withdraw from the project at any time, without giving a reason. If you choose to withdraw during the research project, we will not collect additional information. However, information already collected will be kept and analysed to make sure that the results of the research project can be measured properly. If you do not take part, or withdraw, it will not affect access to the best available treatment options and care from the RCH.

Before deciding whether or not to take part, you might want to talk about the project with a relative, friend or local doctor. Please contact us if you have questions about anything that you don’t understand or want to know more about.

We hope you will take part. If you would like to, please call or email Katherine on the details below.

Principal Investigator Dr Katherine Howell T: (03) 9345 5661 E: [email protected]

If you have any concerns and/or complaints about the project, the way it is being conducted or your rights as a research participant, and would like to speak to someone independent of the project, please contact:

Director, Research Development & Ethics, The Royal Children’s Hospital Melbourne on telephone: (03) 9345 5044.

Version: 7. Date: 07/03/2013 Page 3 of 5 (RDE 07/12: Participant)

CONSENT FORM

HREC Project Number: 32288

Research Project Title: Severe epilepsy in infants

Version Number: 7 Version Date: 7 March 2013

 I have read, or had read to me in my first language, the information statement version listed above and I understand its contents.  I believe I understand the purpose, extent and possible risks of my involvement in this project.  I voluntarily consent to take part in this research project.  I have had an opportunity to ask questions and I am satisfied with the answers I have received.  I understand that this project has been approved by The Royal Children’s Hospital Melbourne Human Research Ethics Committee and will be carried out in line with the National Statement on Ethical Conduct in Human Research (2007).  I understand I will receive a copy of this Information Statement and Consent Form.

OPTIONAL CONSENT

I do I do not consent to my child’s stored DNA sample being used for genetic testing

I do I do not consent to a DNA sample (from blood or cheek swab) being collected from me/us (both parents of the child) to be used for genetic testing to determine the significance of any gene change found in my/our child

I do I do not consent to the storage of my/our and my child’s DNA sample for use in future ethically approved research projects related to epilepsy I wish to be contacted before the sample is used in future research

I do I do not consent to my child’s information and DNA sample being shared with the ‘Genetic Basis of Epilepsy’ (RCH HREC 20114) study

Participant Name Participant Signature Date

Contact Phone Number: ______

The best times to contact me are: ______

Declaration by researcher: I have supplied an Information Letter and Consent Form to the participant who has signed above, and believe that they understand the purpose, extent and possible risks of their involvement in this project.

Research Team Member Name Research Team Member Signature Date Note: All parties signing the Consent Form must date their own signature.

Version: 7. Date: 07/03/2013 Page 4 of 5 (RDE 07/12: Participant)

FORM FOR WITHDRAWAL OF PARTICIPATION

HREC Project Number: 32288

Research Project Title: Severe epilepsy in infants

Version Number: 7 Version Date: 7 March 2013

Declaration by Parent/Guardian

I wish to withdraw from participation in the above research project and understand that such withdrawal will not affect the relationship with those who treated my child or relationship with The Royal Children’s Hospital, Melbourne.

Name of Child (please print)

Name of Parent/Guardian (please print)

Signature of Parent/Guardian Date

Declaration by Study Doctor/Senior Researcher†

I have given a verbal explanation of the implications of withdrawal from the research project and I believe that the Parent/Guardian has understood that explanation.

Name of Study Doctor/ Senior Researcher† (please print)

Signature Date

† A senior member of the research team must provide the explanation of, and information concerning, withdrawal from the research project.

Note: All parties signing the consent section must date their own signature.

Version: 7. Date: 07/03/2013 Page 5 of 5 (RDE 07/12: Participant) Appendix I: Aetiologies of SEI and timing and method of diagnosis in 114 infants

Abbreviations: B = benign, clin = clinical, CMA = chromosomal microarray, D = deleterious, FCD = focal cortical dysplasia, FHx = family history, HIE = hypoxic ischaemic injury, MCD = malformation of cortical development, MIPS = molecular inversion probe-based multigene panel, N/A = not applicable, PCH = pontocerebellar hypoplasia, PD = probably damaging, pre-Sz = before seizure onset, PMG = polymicrogyria, post-Sz = after seizure onset, res gen = research genetic testing, res imag = research imaging review, UK = unknown, USS = ultrasound scan, WES = whole exome sequencing, WGS = whole genome sequencing

411

PATIENT AETIOLOGY DIAGNOSTIC INVESTIGATION

CAUSATIVE (SINGLE) GENE STUDY ID DIAGNOSIS CATEGORY DIAGNOSIS SUSPECTED DIAGNOSIS WHEN DIAGNOSIS MADE HOW DIAGNOSIS MADE IDENTIFIED?

IEE11001 UK UK no N/A N/A N/A IEE11002 UK UK no N/A N/A N/A IEE11003 UK UK no N/A N/A N/A IEE11006 Metabolic PNPO deficiency N/A Post-Sz (clin) Tier 2 PNPO IEE11009 UK UK FCD N/A N/A N/A IEE11010 UK UK no N/A N/A N/A IEE11013 Single gene KCNQ2 encephalopathy N/A Post-Sz (res gen) WES KCNQ2 IEE11015 Metabolic Mitochondrial complex IV deficiency N/A Post-Sz (clin) FHx/clinical/MRI/Tier 2 no IEE11016 Malformative PCH N/A Post-Sz (clin) MRI (Tier 1) no IEE11020 Acquired Complicated meningitis N/A Pre-Sz Clinical/MRI/+ N/A IEE11022 Malformative FCD N/A Post-Sz (clin) Repeat MRI no IEE11026 Chromosomal Del 15q21.3q22.2 N/A Post-Sz (clin) CMA N/A IEE11027 UK UK no N/A N/A N/A IEE11028 Chromosomal Trisomy 21 N/A Pre-Sz Chromosome testing N/A IEE11031 UK UK no N/A N/A N/A IEE11033 Metabolic Molybdenum cofactor deficiency type B N/A Post-Sz (clin) UMS MOCS2 IEE11035 UK UK no N/A N/A N/A IEE11038 UK UK no N/A N/A N/A IEE11041 Acquired HIE N/A Pre-Sz Clinical/MRI/+ N/A IEE12030 Malformative Tuberous sclerosis N/A Pre-Sz Antenatal USS TSC2 IEE12001 UK UK no N/A N/A N/A IEE12002 Single gene SCN8A encephalopathy N/A Post-Sz (clin) Clinical multigene panel SCN8A IEE12003 UK UK FCD N/A N/A N/A IEE12004 Single gene Dravet syndrome N/A Post-Sz (res gen) MIPS SCN1A IEE12005 Acquired HIE and neonatal hypoglycaemia N/A Pre-Sz Clinical/MRI/+ N/A IEE12006 UK UK no N/A N/A N/A IEE12007 Malformative FCD N/A Post-Sz (res imag) MRI (research) DEPDC5 IEE12008 Single gene SCN2A encephalopathy N/A Post-Sz (res gen) WES SCN2A IEE12009 UK UK no N/A N/A N/A IEE12010 Single gene KCNT1 encephalopathy N/A Post-Sz (res gen) MIPS KCNT1 IEE12011 Malformative FCD N/A Post-Sz (clin) Repeat MRI no IEE12012 UK UK no N/A N/A N/A IEE12013 UK UK no N/A N/A N/A IEE12014 Acquired Stroke N/A Pre-Sz Clinical/MRI/+ N/A IEE12015 UK UK no N/A N/A N/A IEE12016 Malformative FCD N/A Post-Sz (clin) Repeat MRI no IEE12017 Malformative FCD N/A Post-Sz (clin) MRI (Tier 1) no IEE12019 Acquired Complicated meningitis N/A Pre-Sz Clinical/MRI/+ N/A IEE12020 Single gene Dravet syndrome N/A Post-Sz (clin) Clinical/single gene SCN1A IEE12021 Chromosomal Trisomy 21 N/A Pre-Sz Chromosome testing N/A IEE12022 Chromosomal Wolf-Hirschhorn syndrome N/A Post-Sz (clin) CMA N/A IEE12023 Single gene Aicardi-Goutieres syndrome N/A Post-Sz (clin) MRI/targeted gene panel RNASEH2B IEE12024 Malformative PMG N/A Post-Sz (clin) MRI (Tier 1) no IEE12025 UK UK no N/A N/A N/A IEE12027 Acquired Prematurity and PVL N/A Pre-Sz Clinical/MRI/+ N/A IEE12028 Malformative FCD N/A Post-Sz (clin) Repeat MRI no IEE12029 Malformative Tuberous sclerosis N/A Pre-Sz Antenatal USS TSC2 IEE12031 Malformative PMG N/A Post-Sz (clin) MRI (Tier 1) no IEE12032 Malformative FCD N/A Post-Sz (clin) MRI (Tier 1) NPRL3 IEE12033 Acquired Prematurity and PVL and IVH N/A Pre-Sz Clinical/MRI/+ N/A IEE12036 UK UK no N/A N/A N/A IEE12037 Single gene SCN2A encephalopathy N/A Post-Sz (res gen) MIPS SCN2A PATIENT AETIOLOGY DIAGNOSTIC INVESTIGATION

CAUSATIVE (SINGLE) GENE STUDY ID DIAGNOSIS CATEGORY DIAGNOSIS SUSPECTED DIAGNOSIS WHEN DIAGNOSIS MADE HOW DIAGNOSIS MADE IDENTIFIED?

IEE12038 UK UK Dravet syndrome N/A N/A N/A IEE12039 Malformative FCD N/A Post-Sz (res imag) MRI (research) no IEE12040 Single gene SMC1A encephalopathy N/A Post-Sz (res gen) WES SMC1A IEE12043 Malformative PMG N/A Post-Sz (clin) MRI no IEE13014 UK UK no N/A N/A N/A IEE13028 Single gene KCNQ2 encephalopathy N/A Post-Sz (res gen) MRI (Tier 1) KCNQ2 IEE13038 Malformative Tuberous sclerosis N/A Pre-Sz Antenatal USS no IEE12045 Acquired HIE and neonatal hypoglycaemia N/A Pre-Sz Clinical/MRI/+ N/A IEE13001 Single gene DOORS syndrome N/A Post-Sz (res gen) Research single gene TBC1D24 IEE13002 Metabolic Mitochondrial N/A Post-Sz (clin) Tier 2 FARS2 (potentially pathogenic) IEE13003 UK UK FCD N/A N/A N/A IEE13004 Malformative MCD (other) N/A Post-Sz (clin) Repeat MRI no IEE13005 UK UK no N/A N/A N/A IEE13006 UK UK no N/A N/A N/A IEE13007 UK UK no N/A N/A N/A IEE13008 Metabolic Mitochondrial complex 1 deficiency N/A Pre-Sz FHx/clinical/single gene yes IEE13009 Single gene SCN8A encephalopathy N/A Post-Sz (res gen) WES SCN8A IEE13010 UK UK no N/A N/A N/A IEE13011 Malformative Achondroplasia N/A Pre-Sz Antenatal USS FGFR3 IEE13012 Chromosomal Trisomy 21 N/A Pre-Sz Chromosome testing N/A IEE13013 Single gene SYNGAP1 encephalopathy N/A Post-Sz (res gen) WGS SYNGAP1 IEE13015 UK UK no N/A N/A N/A IEE13020 Malformative Tuberous sclerosis N/A Post-Sz (clin) Repeat MRI TSC2 IEE13023 Acquired HIE (+? Underlying syndrome) N/A Pre-Sz Clinical/MRI/+ N/A IEE13025 Malformative MCD (other) N/A Pre-Sz MRI no IEE13026 Malformative Aicardi syndrome N/A Pre-Sz MRI/ophthalmology assessment no IEE13027 UK UK no N/A N/A N/A IEE13034 Malformative Lissencephaly N/A Post-Sz (clin) MRI (Tier 1)/CMA N/A IEE13035 Single gene KCNQ2 encephalopathy* N/A Post-Sz (res gen) WES KCNQ2 IEE13036 UK UK FCD N/A N/A N/A IEE13037 Acquired Stroke N/A Pre-Sz Clinical/MRI/+ N/A IEE13039 UK UK no N/A N/A N/A IEE13041 Malformative FCD N/A Post-Sz (clin) Repeat MRI/histopathology no IEE13043 Single gene Sotos syndrome N/A Post-Sz (clin) Clinical/single gene NSD1 IEE13044 UK UK no N/A N/A N/A IEE13045 Malformative Lissencephaly N/A Post-Sz (clin) MRI (Tier 1) no IEE13046 Acquired Prematurity, hypotension, hypoglycaemia N/A Pre-Sz Clinical/MRI/+ N/A IEE13047 UK UK FCD N/A N/A N/A IEE13048 UK UK no N/A N/A N/A IEE14023 UK UK FCD N/A N/A N/A IEE13049 Malformative MCD (other) N/A Post-Sz (clin) MRI (Tier 1) no IEE14001 Malformative FCD N/A Post-Sz (res imag) MRI (research) no IEE14002 Chromosomal Trisomy 21 N/A Pre-Sz Chromosome testing N/A IEE14003 UK UK no N/A N/A N/A IEE14004 Single gene Dravet syndrome N/A Post-Sz (clin) Clinical/single gene SCN1A IEE14006 Malformative FCD N/A Post-Sz (clin) Repeat MRI no IEE14010 Chromosomal Trisomy 21 N/A Pre-Sz Chromosome testing N/A IEE14012 Malformative FCD N/A Post-Sz (clin) Repeat MRI no IEE14013 Acquired HIE N/A Pre-Sz Clinical/MRI/+ N/A IEE14020 Malformative FCD N/A Post-Sz (res imag) MRI (Research) BRAF IEE14026 Chromosomal Isodicentric chr 15 N/A Pre-Sz CMA N/A IEE14027 Malformative FCD N/A Post-Sz (clin) MRI (Tier 1) no PATIENT AETIOLOGY DIAGNOSTIC INVESTIGATION

CAUSATIVE (SINGLE) GENE STUDY ID DIAGNOSIS CATEGORY DIAGNOSIS SUSPECTED DIAGNOSIS WHEN DIAGNOSIS MADE HOW DIAGNOSIS MADE IDENTIFIED?

IEE14030 Chromosomal Del 2q24.3 N/A Post-Sz (clin) CMA N/A IEE14033 UK UK no N/A N/A N/A IEE14038 UK UK no N/A N/A N/A IEE14046 Metabolic Tay-Sachs disease N/A Pre-Sz Tier 2 HEXA IEE14050 Malformative Sturge-Weber syndrome N/A Post-Sz (clin) MRI (Tier 1) no IEE14051 Acquired HIE N/A Pre-Sz Clinical/MRI/+ N/A IEE14015 UK UK no N/A N/A N/A IEE14053 Acquired Ichaemic injury (mechanism unknown) N/A Pre-Sz Clinical/MRI/+ N/A IEE14054 UK UK AARS mutation (biallelic) N/A N/A N/A IEE15009 Malformative Tuberous sclerosis N/A Post-Sz (clin) MRI (Tier 1) TSC1 PATIENT PATHOGENIC/POTENTIALLY PATHOGENIC VARIANTS IDENTIFIED ON RESEARCH GENETIC TESTING IN THIS STUDY

PREVIOUSLY PRESENT IN NORMAL STUDY ID GENE VARIANT REPORTED IN SILICO PREDICTIONS PARENTAL TESTING COMMENT IF PARENTAL TESTING PENDING/NOT DONE or IF VARIANT POTENTIALLY PATHOGENIC DATABASES? PATHOGENIC?

c.545T>G, IEE11013 KCNQ2 p.Val182Gly no no PD (PP2), D (SIFT) pending top-ranked gene clinically c.301G>Q, IEE12004 SCN1A p.Arg101Trp yes no paternal mosaicism c.2390delA, p.Gln797Argfs*18 IEE12007 DEPDC5 (het) no no N/A de novo c.5332A>G, p.Asn1778Asp IEE12008 SCN2A (het) no no PD (PP2), D (SIFT) de novo IEE12010 KCNT1 p.Ala934Thr yes no de novo c.5645G>A, p.Arg1882Gln IEE12037 SCN2A (het) yes no PD (PP2), D (SIFT) de novo c.474delT, p.Ser159ValfsTer1 IEE12040 SMC1A 8 no no N/A pending fits clinically

c.740C>T, top-ranked gene clinically, another non-synonymous variant at same residue is reported pathogenic (S247W, Dedek et al IEE13028 KCNQ2 c.1460dupA,p.Ser247Leu (het) no no PD (PP2), D (SIFT) not done 2003) p.His487Glnfs*71) IEE13001 TBC1D24 / c.313T>C , no no N/A / PD (PP2), D (SIFT) both parents heterozygous

c.195C>A, Fits epilepsy, DD, imaging and biochemical phenotype well, doesn't explain encephalocoele or small kidneys. Postulated to IEE13002 FARS2 (potentially pathogenic) p.Asn65Lys (hmz) no no PD (PP2), D (SIFT) both parents heterozygous have two mendelian disorders c.782G>T, IEE13009 SCN8A p.Cys261Phe no no PD (PP2), D (SIFT) de novo chr6:g.33400507_ De novo (presumed parental gonadal 33400519dup mosaicism as there is a second IEE13013 SYNGAP1 (het) no no N/A affected sibling)

c.793G>A, IEE13035 KCNQ2 p.Ala265Thr (het) yes no D (SIFT), B (PP2) pending top-ranked gene clinically, reported pathogenic 7x (in two publications and in 5x in ClinVar DB)

Appendix J: Classification of seizure types in 114 infants using three versions of the ILAE classification of seizure types

The Clinical Features chapter of this thesis documents the seizure types reported and/or recorded in infants in this study using the 2016 ILAE seizure type classification, with two modifications. This appendix details the seizure types in these infants according to three versions of the ILAE seizure type classification (1981, 2010 and 2016) (Table 1) and then discusses how well and easily the different versions were able to be applied to an infant population.

Seizure types present at onset or evolution of the epilepsy in 114 infants classified using three versions of the ILAE classification of seizure types

2016 classification 2010 classification 1981 classification Unknown – epileptic spasms Unclassified 74 Epileptic spasms 74 74 Focal 61 Partial – simple partial 8 Focal* – motor 49 Partial – complex partial 49 Partial – uncertain 11 Focal – non-motor 24 Partial – partial  GTC 1 Generalised – motor – tonic 15 Generalised – tonic 15 Generalised – tonic 15 Generalised – motor – atonic 1 Generalised – atonic 1 Generalised – atonic 1 Generalised – motor – clonic 0 Generalised – clonic 0 Generalised – clonic 0 Generalised – motor – tonic- Generalised – tonic-clonic 1 Generalised – tonic-clonic 1 clonic 1 Generalised – motor – clonic- tonic-clonic 0 Generalised – motor – Generalised – myoclonic 17 Generalised – myoclonic 17 myoclonic 16 Generalised – motor – myoclonic-atonic 1 Generalised – absence – Generalised – absence Generalised – absence typical 3 (typical) 3 (typical) 4 Generalised – absence – Generalised – absence (with myoclonic 1 special features) 1 Generalised – absence – eyelid myoclonia 0 Generalised – absence – Generalised – absence Generalised – absence atypical 1 (atypical) 1 (atypical) 1 *The reader will note that the number of infants with focal/partial seizures appears to differ across the classifications. In fact, the overall number of infants is the same in each classification (61 infants, which is the number reported using the 2010 classification), some having more than one type of focal seizures. The differences in numbers arise in the 1989 and 2016 classifications, in which subgroups of focal seizure types are present. An infant with more than one type of focal seizures may be listed twice in these classifications if, for example they have both focal – motor and focal – non-motor seizures. Infants with more than one type of seizure in each subgroup (eg two different types of focal – motor seizures with different ictal onsets) were just listed once (ie considered as presence or absence of each seizure type).

417

Discussion The ILAE classifications of seizure types were primarily designed around seizure types seen in older children and adults. Difficulties with their use in infants have been previously described (ref), and were also noted here. We found some difficulties with applying each versions of the classification in this study population, but the two more recent versions were more useful than the 1981 classification. The 1981 classification had two major limitations related to the two most common infant seizure types. Firstly, epileptic spasms are not a classified seizure type; rather they are listed as ‘unclassified’. Secondly, focal (partial) seizures are subclassified according to conscious state during the seizure, which is often difficult to determine in infants. These limitations were overcome in the 2010 classification. Here, all focal seizures are grouped together, without subcategorization according to conscious state, or to motor/non-motor features. In my experience in this study, there was no obvious limitation to that approach. The 2016 classification makes a number of changes relevant to infant seizures. Epileptic spasms can be classified as generalized (generalized – motor – epileptic spasms), focal (focal – motor – epileptic spasms) or of unknown onset (unknown – motor – epileptic spasms). From the description in the classification document, it appears the intended discrimination between focal and generalized is based around the underlying aetiology (eg spasms associated with a focal cortical dysplasia) rather than the electroclinical features of the spasms themselves (eg asymmetric or lateralized spasms clinically or on EEG ictal recording). This appears to be the only seizure type which considers aetiology rather than simply electroclinical features in determining how it is classified. In the 2016 classification, the number of generalized seizure categories has increased, with the addition of clonic-tonic-clonic seizures, myoclonic-atonic seizures (previously in the generalized – myoclonic group), myoclonic absences and absences with eyelid myoclonia (previously in ‘generalised - absence (with special features)’). It is difficult to comment on whether this is useful from the experience of this study, given relatively few infants had myoclonic and absence seizures (of any subtype). Subclassifications of focal seizures have been reinstated in the 2016 classification. Here, focal seizures are subclassified as motor or non-motor, further characterized by more detailed semiology (eg clonic or autonomic), and with an additional descriptor of awareness. This reintroduces the issue of accurate determination of conscious state in infants. It produces fairly detailed focal seizure subgroups, some of which are not easy to apply or commonly seen in infants, such as ‘sensory’, ‘emotional’ and ‘hypermotor’. Overall, the simplicity (and fewer subcategories) of the 2010 classification was the easiest to apply to the infant population.

418

Appendix K: Epileptic syndrome classification in 114 infants with SEI

Abbreviations:

Other: Hx = history, N/A = not applicable, Rx = treatment

Seizure types: ES = epileptic spasms, F-M = focal motor seizures, F-NM = focal non-motor seizures, G- Ab(atyp) = generalised atypical absence seizures, G-Ab(myo) = generalised myoclonic absence seizures, G- Ab(typ) = generalised typical absence seizures, G-At = generalised atonic seizures, G-myo = generalised myoclonic seizures, G-Myo-At = generalised myoclonic-atonic seizures, G-T = generalised tonic seizures

Interictal EEG: B-G = generalised background abnormality (slowing), F-uni = unifocal, F-multi = multifocal, G- SW +/- PFA = generalised spike-wave +/- paroxysmal fast activity, G-hyps = hypsarrhythmia, G-BS = generalised burst-suppression, N = normal, Mix = generalised and focal epileptiform discharges

Ictal EEG: F-uni = unifocal, F-multi = multifocal, G-combo = a number of generalised seizure patterns, G-EDR = generalised electrodecrement, G-Sp = spasm complexes, G-SW = generalised spike-wave

Epileptic syndromes (see Methods chapter for syndrome definitions): BMEI-like = benign myoclonic epilepsy of infancy-like, CAE-like = childhood absence epilepsy-like, DS = Dravet syndrome, DS-like = Dravet syndrome-like, EIEE = early infantile epileptic encephalopathy (Ohtahara syndrome), EIEE-like = early infantile epileptic encephalopathy-like, EIEE-plus = early infantile epileptic encephalopathy-plus, EIMFS = epilepsy of infancy with migrating focal seizures, EIMFS-like = epilepsy of infancy with migrating focal seizures-like, EIMFS-plus = epilepsy of infancy with migrating focal seizures-plus, EME = early myoclonic encephalopathy, EME-like = early myoclonic encephalopathy-plus, Focal (multi) = multifocal epilepsy, Focal (multi) = multifocal epilepsy, Focal (uni) = unifocal epilepsy, LGS = Lennox Gastaut syndrome, LGS-like = Lennox Gastaut syndrome-like, MAE-like = epilepsy with myoclonic-atonic seizures-like, WS = West syndrome, WS-like = West syndrome-like, WS-like+ = West syndrome-like-plus, WS-+ = West syndrome-plus

419

Onset Evolution Treatment Ongoing Time to Age of seizure seizures? (at 2 ID presentation Living? onset (months) years old or (months) Non-pharm- Epileptic Epileptic death) Based on Seizure types Interictal EEG Ictal EEG Difference from prototypic syndrome Present? Based on Seizure types Interictal EEG Ictal EEG Difference from prototypic syndrome Number of AEDs acologic syndrome syndrome treatment

IEE11001 3 <1 ictal EEG + video +Hx + interictal EEG F-M F-multi F-uni Focal (other) N/A no 3 nil yes yes

G-Ab(myo), G- IEE11002 2 <1 video + Hx + interictal EEG F-M G-SW+/-PFA DS-like Later seizure types not present and early onset yes ictal EEG + video +Hx + interictal EEG Mix Mix DS-like Early onset, prominent focal IEDs (in addition to GSW) 6 nil yes yes Myo, F-M IEE11003 7 <1 Hx +interictal EEG ES G-hyps WS N/A no 1 nil no (off Rx) yes

IEE11006 0 <1 ictal EEG + video +Hx + interictal EEG G-Myo G-BS G-SW EME N/A yes ictal EEG + video +Hx + interictal EEG G-Myo, G-T B-G G-combo LGS-like No slow spike-wave 5 nil no (off Rx) yes

IEE11009 2 <2 video + Hx + interictal EEG F-M F-multi Focal (other) N/A yes ictal EEG + video +Hx + interictal EEG ES G-hyps G-Sp WS N/A 4 nil no (on Rx) yes

IEE11010 6 <1 Hx +interictal EEG ES G-hyps WS N/A no 3 nil no (off Rx) yes

IEE11013 0 <1 ictal EEG + video +Hx + interictal EEG F-M, F-NM G-BS F-multi EIEE-like Tonic seizures focal no 5 nil no (on Rx) yes

IEE11015 0 <3 Hx +interictal EEG F-M, F-NM, G-T G-BS EIEE+ Additional seizure types (focal) no 7 nil yes yes

IEE11016 0 <1 Hx +interictal EEG F-M, F-NM F-multi Focal (other) N/A yes ictal EEG + video +Hx + interictal EEG ES G-hyps G-Sp WS N/A 6 nil yes yes

IEE11020 3 <1 video + Hx + interictal EEG ES G-hyps WS N/A yes Hx +interictal EEG ES, F-M F-multi WS-like+ No hypsarrhythmia, additional seizure type (focal) 3 nil yes yes

IEE11022 6 <1 ictal EEG + video +Hx + interictal EEG ES G-hyps G-Sp WS N/A yes ictal EEG + video +Hx + interictal EEG ES F-uni F-uni WS-like No hypsarrhythmia 4 nil yes yes

Pharmacoresistant, ictal EEG bicentral spike-wave (ie IEE11026 3 <1 ictal EEG + video +Hx + interictal EEG G-Myo N F-multi BMEI-like no 3 nil no (off Rx) yes regionalised rather than generalised spike-wave) IEE11027 2 <1 ictal EEG + video +Hx + interictal EEG F-M F-multi F-multi EIMFS N/A yes Hx +interictal EEG ES, F-M G-hyps WS+ Additional seizure type (focal) 6 nil yes no

IEE11028 8 <1 ictal EEG + video +Hx + interictal EEG ES G-hyps G-Sp WS N/A no 1 nil no (off Rx) yes

IEE11031 0 <1 ictal EEG + video +Hx + interictal EEG F-M G-BS F-multi EIEE-like Tonic seizures focal no 4 nil yes no

IEE11033 0 <1 ictal EEG + video +Hx + interictal EEG F-M G-BS F-multi EIEE-like N/A yes video + Hx + interictal EEG ES B-G WS-like No hypsarrhythmia 5 nil yes yes

IEE11035 6 <1 Hx +interictal EEG F-M F-multi Focal (other) N/A no 5 nil yes no

IEE11038 2 <1 ictal EEG + video +Hx + interictal EEG F-M, F-NM F-multi F-multi EIMFS N/A yes ictal EEG + video +Hx + interictal EEG ES, G-Myo B-G G-Sp WS-like+ No hypsarrhythmia, additional seizure type (myoclonic) 13 Modified Atkins Diet yes yes

IEE11041 1 <1 Hx +interictal EEG ES G-hyps WS N/A no 6 Keto diet no (on Rx) yes

IEE12030 1 <2 Hx +interictal EEG F-M, F-NM F-multi Focal (multi) N/A yes ictal EEG + video +Hx + interictal EEG ES, F-M, F-NM G-hyps G-Sp WS+ Additional seizure type (focal) 6 surgery no (on Rx) yes

IEE12001 1 <1 ictal EEG + video +Hx + interictal EEG F-M F-multi F-uni Focal (other) N/A yes ictal EEG + video +Hx + interictal EEG ES, G-T, G-Myo F-multi G-combo LGS-like No slow spike-wave 13 Keto diet yes yes

IEE12002 1 <1 ictal EEG + video +Hx + interictal EEG F-M, F-NM F-multi F-uni EIMFS-like Independent left and right seizure onsets not confirmed no 6 nil yes no

IEE12003 6 <1 ictal EEG + video +Hx + interictal EEG ES G-hyps G-Sp WS N/A no 1 nil no (off Rx) yes

F-M, G-Ab(typ), IEE12004 5 <2 video + Hx + interictal EEG F-M N DS-like Later seizure types not present yes ictal EEG + video +Hx + interictal EEG G-SW+/-PFA G-SW DS N/A 6 nil yes yes G-Myo IEE12005 6 <1 ictal EEG + video +Hx + interictal EEG ES G-hyps G-Sp WS N/A yes ictal EEG + video +Hx + interictal EEG G-T F-multi G-EDR LGS-like No slow spike-wave 7 nil yes yes

IEE12006 1 <1 ictal EEG + video +Hx + interictal EEG F-M, F-NM F-multi F-multi EIMFS N/A yes ictal EEG + video +Hx + interictal EEG ES, F-M G-hyps G-Sp WS+ Additional seizure type 11 nil yes yes

IEE12007 2 <2 ictal EEG + video +Hx + interictal EEG ES F-uni G-Sp WS-like No hypsarrhythmia yes ictal EEG + video +Hx + interictal EEG F-NM F-uni F-uni Focal (uni) N/A 5 nil yes yes

IEE12008 0 <1 ictal EEG + video +Hx + interictal EEG F-M G-BS F-multi EIEE-like Tonic seizures focal no 6 nil yes no

IEE12009 3 <1 ictal EEG + video +Hx + interictal EEG ES G-hyps G-Sp WS N/A no 7 Keto diet yes yes

IEE12010 0 <1 ictal EEG + video +Hx + interictal EEG F-M F-multi F-multi EIMFS N/A no 7 nil yes yes

IEE12011 2 <1 ictal EEG + video +Hx + interictal EEG ES F-uni G-Sp WS-like No hypsarrhythmia no 1 nil no (off Rx) yes

IEE12012 4 <1 ictal EEG + video +Hx + interictal EEG ES, G-Myo G-hyps G-combo WS+ Additional seizure type (myoclonic) yes ictal EEG + video +Hx + interictal EEG ES, F-M F-multi F-multi WS-like+ No hypsarrhythmia, additional seizure type (focal) 6 nil yes yes

IEE12013 17 <1 Hx +interictal EEG ES G-hyps WS N/A no 2 nil no (off Rx) yes

IEE12014 5 <7 ictal EEG + video +Hx + interictal EEG ES, G-Myo G-hyps G-combo WS+ Additional seizure type (myoclonic) no 2 nil no (on Rx) yes

IEE12015 3 <6 ictal EEG + video +Hx + interictal EEG ES G-hyps G-Sp WS N/A yes ictal EEG + video +Hx + interictal EEG ES, G-T F-multi G-EDR LGS-like No slow spike-wave 7 nil yes yes

IEE12016 11 <1 ictal EEG + video +Hx + interictal EEG F-M F-uni F-uni Focal (uni) N/A no 6 nil yes yes

IEE12017 5 <3 Hx +interictal EEG ES G-hyps WS N/A yes Hx +interictal EEG ES F-uni WS-like No hypsarrhythmia 2 nil yes yes

IEE12019 11 <1 Hx +interictal EEG F-NM F-uni Focal (uni) N/A yes Hx +interictal EEG ES G-hyps WS N/A 2 nil yes yes

F-M, G-Ab(typ), IEE12020 6 <1 Hx +interictal EEG F-M G-SW+/-PFA DS-like Later seizure types not present yes Hx +interictal EEG G-SW+/-PFA DS N/A 2 nil yes yes G-Myo IEE12021 5 <5 Hx +interictal EEG ES G-hyps WS N/A no 3 nil no (on Rx) yes

IEE12022 0 <1 video + Hx + interictal EEG G-Myo F-multi EME-like No burst-suppression (discontinuous only) no 4 nil yes no

IEE12023 10 <1 Hx +interictal EEG F-M F-multi Focal (other) N/A no 4 nil no (on Rx) yes

IEE12024 0 <1 ictal EEG + video +Hx + interictal EEG F-NM F-multi F-multi EIMFS N/A no 8 nil yes no

IEE12025 7 <2 video + Hx + interictal EEG ES No EEG WS N/A no 0 nil no (off Rx) yes

IEE12027 12 <1 ictal EEG + video +Hx + interictal EEG G-Ab(typ) F-multi G-SW CAE-like Early onset no 2 nil yes yes

IEE12028 4 <1 ictal EEG + video +Hx + interictal EEG ES F-uni G-Sp WS-like No hypsarrhythmia no 8 surgery no (off Rx) yes

IEE12029 3 <1 ictal EEG + video +Hx + interictal EEG ES G-SW+/-PFA G-Sp WS-like No hypsarrhythmia yes ictal EEG + video +Hx + interictal EEG ES, F-NM F-multi Mix WS-like+ No hypsarrhythmia, additional seizure type (focal) 8 nil no (on Rx) yes

IEE12031 1 <1 ictal EEG + video +Hx + interictal EEG ES, F-M F-uni G-Sp WS-like+ No hypsarrhythmia, additional seizure type (focal) 1 nil no (off Rx) yes

IEE12032 0 <1 ictal EEG + video +Hx + interictal EEG F-M F-uni F-uni Focal (uni) N/A yes Hx +interictal EEG ES, F-NM F-multi F-uni WS-like+ No hypsarrhythmia, additional seizure type (focal) 4 surgery no (on Rx) yes IEE12033 7 <1 ictal EEG + video +Hx + interictal EEG ES G-hyps G-Sp WS N/A no 3 nil no (off Rx) yes

IEE12036 1 <1 ictal EEG + video +Hx + interictal EEG G-Myo G-SW+/-PFA G-SW MAE-like Abnormal development before seizure onset no 3 nil yes no

IEE12037 0 <1 ictal EEG + video +Hx + interictal EEG F-M, F-NM G-BS F-multi EIEE-like Tonic seizures focal yes ictal EEG + video +Hx + interictal EEG G-T G-hyps Nil LGS-like No slow spike-wave 6 nil yes no

IEE12038 4 <2 Hx +interictal EEG F-M N DS-like Later seizure types not present yes ictal EEG + video +Hx + interictal EEG F-M, G-Myo B-G G-SW DS N/A 5 nil yes yes

IEE12039 5 <2 Hx only F-NM No EEG Focal (uni) N/A yes video + Hx + interictal EEG ES G-hyps WS N/A 3 nil no (on Rx) yes

IEE12040 6 <2 ictal EEG + video +Hx + interictal EEG ES, F-M G-hyps F-uni WS+ Additional seizure type (focal) no 8 nil yes yes

IEE12043 3 <1 Hx +interictal EEG ES F-multi WS-like No hypsarrhythmia yes ictal EEG + video +Hx + interictal EEG ES, G-T, G-Myo G-SW+/-PFA G-SW LGS-like No tonic seizures 6 nil yes yes

IEE13014 2 <1 Hx +interictal EEG F-M F-multi Focal (other) N/A no 4 nil yes no

IEE13028 0 <1 ictal EEG + video +Hx + interictal EEG F-M F-multi F-multi EIMFS N/A no 7 Keto diet no (on Rx) yes

IEE13038 2 <1 ictal EEG + video +Hx + interictal EEG F-M F-uni F-uni Focal (uni) N/A yes ictal EEG + video +Hx + interictal EEG ES, F-M G-hyps G-EDR WS+ Additional seizure type (focal) 6 nil yes yes

IEE12045 1 <1 Hx +interictal EEG F-M N DS-like Later seizure types not present yes ictal EEG + video +Hx + interictal EEG ES G-hyps G-EDR WS N/A 1 nil yes yes

IEE13001 0 <2 ictal EEG + video +Hx + interictal EEG ES, F-M, F-NM F-multi Mix EIMFS+ Additional seizure type (spasms) no 7 Keto diet yes no

IEE13002 1 <1 ictal EEG + video +Hx + interictal EEG F-NM F-multi F-multi EIMFS N/A no 7 Keto diet yes no

IEE13003 3 <4 Hx +interictal EEG ES G-hyps WS N/A no 1 nil no (off Rx) yes

IEE13004 3 <4 ictal EEG + video +Hx + interictal EEG ES G-hyps G-Sp WS N/A yes video + Hx + interictal EEG ES G-SW+/-PFA G-Sp LGS-like No tonic seizures 4 nil yes yes

IEE13005 6 <1 Hx +interictal EEG ES G-hyps WS N/A yes ictal EEG + video +Hx + interictal EEG ES G-SW+/-PFA G-Sp LGS-like No tonic seizures 6 Keto diet yes yes

IEE13006 7 <1 ictal EEG + video +Hx + interictal EEG ES G-hyps G-Sp WS N/A no 2 nil no (off Rx) yes

IEE13007 3 <1 Hx +interictal EEG F-M F-multi Focal (multi) N/A yes ictal EEG + video +Hx + interictal EEG ES, F-M G-hyps Mix WS+ Additional seizure type (focal) 4 nil no (on Rx) yes

IEE13008 16 <3 ictal EEG + video +Hx + interictal EEG F-M B-G F-uni Focal (uni) N/A yes Hx +interictal EEG ES, F-M WS+ Additional seizure type (focal) 3 nil yes yes

IEE13009 7 <1 ictal EEG + video +Hx + interictal EEG F-M F-multi F-uni Focal (other) N/A no 3 nil no (on Rx) yes

IEE13010 7 <1 ictal EEG + video +Hx + interictal EEG ES G-hyps G-Sp WS N/A no 1 nil no (off Rx) yes

IEE13011 3 <1 ictal EEG + video +Hx + interictal EEG F-NM F-multi F-multi Focal (multi) N/A no 4 nil no (on Rx) yes

IEE13012 4 <1 ictal EEG + video +Hx + interictal EEG ES G-hyps G-Sp WS N/A no 1 nil no (off Rx) yes

IEE13013 11 <9 Hx only G-Myo No EEG MAE-like Abnormal development before seizure onset yes ictal EEG + video +Hx + interictal EEG G-Myo, G-At G-SW+/-PFA G-SW MAE-like Abnormal development before seizure onset 2 Modified Atkins Diet yes yes

IEE13015 0 <1 ictal EEG + video +Hx + interictal EEG G-T G-BS G-EDR EIEE N/A no 1 nil yes no

IEE13020 12 <2 Hx only F-NM No EEG Focal (uni) N/A yes Hx +interictal EEG ES, F-NM G-hyps WS+ Additional seizure type (focal) 3 nil yes yes

IEE13023 9 <1 ictal EEG + video +Hx + interictal EEG G-T, F-M F-multi Mix LGS-like No slow spike-wave, onset < 1 year no 3 nil yes yes

IEE13025 10 <1 Hx +interictal EEG ES, F-M F-uni WS-like+ Additional seizure type (focal) no 2 nil no (on Rx) yes

IEE13026 6 <1 video + Hx + interictal EEG ES F-multi WS-like No hypsarrhythmia no 2 nil no (on Rx) no

IEE13027 5 <2 ictal EEG + video +Hx + interictal EEG ES G-hyps G-Sp WS N/A no 1 nil no (off Rx) yes

IEE13034 4 <1 Hx +interictal EEG ES G-hyps WS N/A no 4 nil no (on Rx) yes

IEE13035 0 <5 Hx +interictal EEG G-T G-BS EIEE N/A no 3 nil no (on Rx) yes

IEE13036 4 <1 ictal EEG + video +Hx + interictal EEG ES F-uni G-Sp WS-like No hypsarrhythmia no 1 nil no (off Rx) yes

IEE13037 11 <2 video + Hx + interictal EEG ES Mix WS-like No hypsarrhythmia no 3 nil no (on Rx) yes

IEE13039 12 <3 ictal EEG + video +Hx + interictal EEG ES G-hyps G-Sp WS N/A yes ictal EEG + video +Hx + interictal EEG ES, G-T G-EDR LGS-like No slow spike-wave 4 nil no (on Rx) yes

IEE13041 9 <1 ictal EEG + video +Hx + interictal EEG ES G-hyps G-Sp WS N/A yes ictal EEG + video +Hx + interictal EEG ES, G-T G-SW+/-PFA G-combo LGS N/A 7 nil yes yes

IEE13043 7 <1 Hx +interictal EEG ES G-hyps WS N/A no 4 nil no (off Rx) yes

IEE13044 5 <1 ictal EEG + video +Hx + interictal EEG F-M, F-NM F-multi F-multi EIMFS N/A no 8 nil yes no

IEE13045 2 <1 video + Hx + interictal EEG F-M F-multi Focal (other) N/A no 4 nil yes yes

IEE13046 8 <1 ictal EEG + video +Hx + interictal EEG ES G-hyps G-Sp WS N/A no 3 nil no (on Rx) yes

IEE13047 16 <1 Hx only F-M No EEG Focal (uni) N/A yes ictal EEG + video +Hx + interictal EEG ES, F-M F-uni Mix WS-like+ No hypsarrhythmia, additional seizure type (focal) 3 nil no (on Rx) yes

IEE13048 2 >12 ictal EEG + video +Hx + interictal EEG ES G-hyps G-Sp WS N/A no 2 nil no (off Rx) yes

IEE14023 3 <3 Hx +interictal EEG ES G-hyps WS N/A yes ictal EEG + video +Hx + interictal EEG ES, G-T, G-Myo G-EDR+/-PFA G-combo LGS-like No slow spike-wave 2 nil no (on Rx) yes

IEE13049 0 <1 Hx +interictal EEG F-M F-multi Focal (other) N/A no 5 nil yes no

IEE14001 14 <2 ictal EEG + video +Hx + interictal EEG ES G-hyps G-Sp WS N/A no 1 nil no (off Rx) yes

IEE14002 5 <2 video + Hx + interictal EEG ES G-hyps WS N/A no 1 nil no (off Rx) yes

IEE14003 4 <4 Hx only F-M No EEG Focal (multi) N/A yes ictal EEG + video +Hx + interictal EEG ES, F-M G-hyps Mix WS+ Additional seizure type (focal) 3 nil no (on Rx) yes

IEE14004 4 <1 Hx +interictal EEG F-M N DS-like Later seizure types not present yes video + Hx + interictal EEG F-M G-SW+/-PFA DS N/A 5 nil yes yes

IEE14006 5 <1 ictal EEG + video +Hx + interictal EEG F-M F-uni F-uni Focal (uni) N/A yes ictal EEG + video +Hx + interictal EEG ES, F-M G-hyps Mix WS+ Additional seizure type (focal) 8 surgery yes yes

IEE14010 5 <1 ictal EEG + video +Hx + interictal EEG ES G-hyps G-Sp WS N/A no 2 nil no (off Rx) yes

IEE14012 2 <1 Hx +interictal EEG F-NM F-uni Focal (uni) N/A yes ictal EEG + video +Hx + interictal EEG ES, F-NM G-hyps G-Sp WS+ Additional seizure type (focal) 5 surgery no (off Rx) yes

IEE14013 8 <2 Hx +interictal EEG ES G-hyps WS N/A no 4 nil no (on Rx) yes

IEE14020 9 <2 ictal EEG + video +Hx + interictal EEG F-M F-uni F-uni Focal (uni) N/A no 3 nil yes no Onset Evolution Treatment Ongoing Time to Age of seizure seizures? (at 2 ID presentation Living? onset (months) years old or (months) Non-pharm- Epileptic Epileptic death) Based on Seizure types Interictal EEG Ictal EEG Difference from prototypic syndrome Present? Based on Seizure types Interictal EEG Ictal EEG Difference from prototypic syndrome Number of AEDs acologic syndrome syndrome treatment

ES, G-T, G- IEE14026 9 <1 Hx +interictal EEG ES G-hyps WS N/A yes ictal EEG + video +Hx + interictal EEG G-SW+/-PFA G-combo LGS-like No slow spike-wave 3 nil yes yes Ab(atyp) IEE14027 13 <6 Hx +interictal EEG ES G-hyps WS N/A no 2 nil no (on Rx) yes

Later seizure types not present, early onset, abnormal devt IEE14030 3 <1 Hx +interictal EEG F-M F-multi DS-like yes Hx +interictal EEG F-M, F-NM, G-Myo DS-like Abnormal development before seizure onset 7 nil yes no prior to Sz onset IEE14033 12 <4 ictal EEG + video +Hx + interictal EEG F-M F-multi F-multi Focal (multi) N/A yes Hx +interictal EEG ES, F-M F-multi WS-like+ No hypsarrhythmia, additional seizure type (focal) 6 nil yes yes

IEE14038 17 <1 video + Hx + interictal EEG F-M, F-NM F-uni Focal (other) N/A no 4 nil yes yes

IEE14046 14 <1 ictal EEG + video +Hx + interictal EEG G-T, F-NM F-multi G-EDR LGS-like No slow spike-wave no 2 nil yes yes

IEE14050 9 <1 ictal EEG + video +Hx + interictal EEG F-NM F-uni F-uni Focal (uni) N/A yes ictal EEG + video +Hx + interictal EEG G-Myo-At Mix G-SW MAE-like Abnormal MRI brain 7 nil yes yes

IEE14051 3 <1 ictal EEG + video +Hx + interictal EEG ES, F-M F-multi Mix WS-like+ No hypsarrhythmia, additional seizure type (focal) no 4 nil yes yes

IEE14015 4 <3 ictal EEG + video +Hx + interictal EEG G-T F-multi G-EDR LGS-like No slow spike-wave, onset < 1 year no 1 nil no (off Rx) yes

IEE14053 12 <1 ictal EEG + video +Hx + interictal EEG ES G-hyps G-Sp WS N/A no 5 nil yes yes

IEE14054 8 <7 ictal EEG + video +Hx + interictal EEG ES G-hyps WS N/A yes Hx +interictal EEG G-Myo G-SW+/-PFA MAE-like Abnormal development before seizure onset 3 nil yes yes

IEE15009 6 <11 Hx only ES No EEG WS N/A yes ictal EEG + video +Hx + interictal EEG G-ES, F-NM F-uni F-uni WS-like+ No hypsarrhythmia, additional seizure type (focal) 2 nil yes yes Appendix L: Examples of how prototypic and variant epileptic syndromes were assigned

423

Figure 1: EEGs in infant IEE13015 with EIEE (A and B) shown using a neonatal montage, and IEE11013 with an EIEE-like phenotype (C and D) shown using an AP bipolar montage. Interictal EEGs (A and C) show burst-suppression. Ictal EEGs during tonic seizures show a generalised ictal rhythm (B) and a focal ictal rhythm over the right temporal region (D) A B

C D

Figure 2: EEGs in infant IEE13036 with WS (A and B) and IEE11022 with an WS-like phenotype (C and D) showing using an AP bipolar montage. Interictal EEGs show modified hypsarrhythmia (A) and unifocal epileptiform discharges (C). Ictal EEGs during spasms (B and D) show periodic spasm complexes A B

C D Figure 3: EEGs in infant IEE12012 with a WS-plus phenotype (A-C) and IEE15009 with an WS-like-plus phenotype (D-F) shown using an AP bipolar montage unless stated. Interictal EEGs show modified hypsarrhythmia (A) and unifocal epileptiform discharges (D). Ictal EEGs during spasms (B and E) show periodic spasm complexes (note transverse montage in E). Ictal EEGs of the infants’ additional seizure types show generalised spike-wave during a myoclonic jerk (C) and a focal rhythm over the right temporal region during a focal seizure (F)

A D

B E

C F

Appendix M: Patient data and investigation costs used to model diagnostic pathways

Appendix shows data on individual patients that was used to simulate how and when aetiologic diagnosis was made, followed by costs incurred in diagnostic investigation. These two parts of the appendix do not include the confirmatory costs for suspected diagnoses made on tests in the diagnostic pathway that needed confirmation with genetic testing or mitochondrial respiratory enzyme chain analysis on liver/muscle. The final part of this appendix shows the group level costs (including the costs for confirmatory testing of suspected diagnoses where relevant). Most group level data is also included in the Diagnostic Investigation chapter.

427

PATIENT DATA

Dx made Tier Sz ongoing Sz ongoing Sz ongoing 1 *USE THIS >1m (If no Dx >3m (If no Dx >6m (If no Dx Dx made Dx known pre- Tier 1 testing Dx made Tier COLUMN IF Tier 2 testing Dx made Tier Rpt MRI Dx made Rpt Dx made Tier Multigene STUDY ID made after made after made after Tier 3 done? multigene Sz done? 1 MULTIGENE done? 2 done? MRI 3 testing done? Tier 1 testing Tier 2 testing rpt MRI or if testing TESTING STEP or if pt died) or if pt died) pt died) 2

IEE11001 0 1 0 0 1 1 0 1 1 0 1 1 0 1 0 IEE11002 0 1 0 0 1 1 0 1 1 0 1 1 0 1 0 IEE11003 0 1 0 0 0 1 0 0 1 0 0 0 0 1 0 IEE11006 0 1 0 0 1 1 1 1 N/A 0 1 N/A N/A 1 1 IEE11009 0 1 0 0 1 1 0 1 1 0 0 0 0 1 0 IEE11010 0 1 0 0 1 1 0 0 1 0 0 0 0 1 0 IEE11013 0 1 0 0 1 1 0 0 1 0 0 1 0 1 1 IEE11015 0 1 0 0 1 1 1 1 N/A 0 1 N/A N/A 1 0 IEE11016 0 1 1 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A 1 0 IEE11020 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE11022 0 1 0 0 1 1 0 1 1 1 1 N/A N/A 1 0 IEE11026 0 1 1 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A 1 0 IEE11027 0 1 0 0 1 1 0 1 1 0 1 1 0 1 0 IEE11028 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE11031 0 1 0 0 1 1 0 1 1 0 1 1 0 1 0 IEE11033 0 1 1 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A 1 1 IEE11035 0 1 0 0 1 1 0 1 1 0 1 1 0 1 0 IEE11038 0 1 0 0 1 1 0 1 1 0 1 1 0 1 0 IEE11041 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE12030 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE12001 0 1 0 0 1 1 0 1 1 0 1 1 0 1 0 IEE12002 0 1 0 0 1 1 0 1 1 0 1 1 0 1 1 IEE12003 0 1 0 0 0 1 0 0 1 0 0 0 0 1 0 IEE12004 0 1 1 0 1 N/A N/A N/A N/A N/A N/A N/A N/A 1 1 IEE12005 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE12006 0 1 0 0 1 1 0 1 1 0 1 1 0 1 0 IEE12007 0 1 0 0 1 1 0 1 1 1 1 N/A N/A 1 1 IEE12008 0 1 0 0 1 1 0 1 1 0 1 1 0 1 1 IEE12009 0 1 0 0 1 1 0 1 1 0 1 1 0 1 0 IEE12010 0 1 0 0 1 1 0 1 1 0 1 1 0 1 1 IEE12011 0 1 0 0 0 1 0 0 1 1 0 N/A N/A 1 0 IEE12012 0 1 0 0 1 1 0 1 1 0 1 1 0 1 0 IEE12013 0 1 0 0 0 1 0 0 1 0 0 0 0 1 0 IEE12014 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE12015 0 1 0 0 1 1 0 1 1 0 1 1 0 1 0 IEE12016 0 1 0 0 1 1 0 1 1 1 1 N/A N/A 1 0 IEE12017 0 1 1 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A 1 0 IEE12019 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE12020 0 1 1 0 1 N/A N/A N/A N/A N/A N/A N/A N/A 1 1 IEE12021 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE12022 0 1 1 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A 1 0 IEE12023 0 1 1 0 1 N/A N/A N/A N/A N/A N/A N/A N/A 1 1 IEE12024 0 1 1 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A 1 0 IEE12025 0 1 0 0 0 1 0 0 1 0 0 0 0 1 0 IEE12027 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE12028 0 1 0 0 1 1 0 1 1 1 1 N/A N/A 1 0 IEE12029 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE12031 0 1 1 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A 1 0 IEE12032 0 1 1 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A 1 1 IEE12033 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE12036 0 1 0 0 1 1 0 1 1 0 1 1 0 1 0 IEE12037 0 1 0 0 1 1 0 1 1 0 1 1 0 1 1 IEE12038 0 1 1 0 1 N/A N/A N/A N/A N/A N/A N/A N/A 1 1 IEE12039 0 1 1 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A 1 0 IEE12040 0 1 0 0 1 1 0 1 1 0 1 1 0 1 1 IEE12043 0 1 1 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A 1 0 IEE13014 0 1 0 0 1 1 0 1 1 0 1 1 0 1 0 IEE13028 0 1 0 0 1 1 0 1 1 0 0 1 0 1 1 IEE13038 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE12045 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE13001 0 1 1 0 1 N/A N/A N/A N/A N/A N/A N/A N/A 1 1 IEE13002 0 1 0 0 1 1 1 1 N/A 0 1 N/A N/A 1 0 IEE13003 0 1 0 0 0 1 0 0 1 0 0 0 0 1 0 IEE13004 0 1 0 0 1 1 0 1 1 1 1 1 0 1 0 IEE13005 0 1 0 0 1 1 0 1 1 0 1 1 0 1 0 IEE13006 0 1 0 0 0 1 0 0 1 0 0 0 0 1 0 IEE13007 0 1 0 0 1 1 0 1 1 0 0 0 0 1 0 IEE13008 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE13009 0 1 0 0 1 1 0 0 1 0 0 0 0 1 1 IEE13010 0 1 0 0 0 1 0 0 1 0 0 0 0 1 0 IEE13011 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE13012 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE13013 0 1 0 0 1 1 0 1 1 0 1 1 0 1 1 IEE13015 0 1 0 0 1 1 0 1 1 0 1 1 0 1 0 IEE13020 0 1 1 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A 1 1 IEE13023 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE13025 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE13026 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE13027 0 1 0 0 0 1 0 0 1 0 0 0 0 1 0 IEE13034 0 1 1 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A 1 1 IEE13035 0 1 0 0 0 1 0 0 1 0 0 1 0 1 1 IEE13036 0 1 0 0 0 1 0 0 1 0 0 0 0 1 0 IEE13037 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE13039 0 1 0 0 1 1 0 1 1 0 1 0 0 1 0 IEE13041 0 1 0 0 1 1 0 1 1 1 1 N/A N/A 1 0 IEE13043 0 1 1 0 1 N/A N/A N/A N/A N/A N/A N/A N/A 1 1 IEE13044 0 1 0 0 1 1 0 1 1 0 1 1 0 1 0 IEE13045 0 1 1 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A 1 1 IEE13046 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE13047 0 1 0 0 1 1 0 1 1 0 1 0 0 1 0 IEE13048 0 1 0 0 1 1 0 0 1 0 0 0 0 1 0 IEE14023 0 1 0 0 0 1 0 0 1 0 0 0 0 1 0 IEE13049 0 1 1 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A 1 0 IEE14001 0 1 1 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A 1 0 IEE14002 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE14003 0 1 0 0 1 1 0 1 1 0 0 0 0 1 0 IEE14004 0 1 1 0 1 N/A N/A N/A N/A N/A N/A N/A N/A 1 1 IEE14006 0 1 0 0 1 1 0 1 1 1 1 N/A N/A 1 0 IEE14010 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE14012 0 1 0 0 1 1 0 1 1 1 1 N/A N/A 1 0 IEE14013 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE14020 0 1 1 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A 1 1 IEE14026 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE14027 0 1 1 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A 1 0 IEE14030 0 1 1 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A 1 0 IEE14033 0 1 0 0 1 1 0 1 1 0 1 1 0 1 0 IEE14038 0 1 0 0 1 1 0 1 1 0 1 1 0 1 0 IEE14046 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE14050 0 1 1 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A 1 0 IEE14051 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE14015 0 1 0 0 0 1 0 0 1 0 0 0 0 1 0 IEE14053 1 N/A N/A 0 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IEE14054 0 1 0 0 1 1 0 1 1 0 1 0 0 1 0 IEE15009 0 1 1 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A 1 1 SCENARIOS IN WHICH ALL INFANTS CONTINUE THROUGH DIAGNOSTIC PATHWAY Scenario 1 (No multigene) Scenario 2 (multigene pos 5)

Diagnosis Diagnosis Tier 1 Testing Tier 2 Testing Tier 3 Testing made Tier 1 Testing Tier 2 Testing Tier 3 Testing Multigene made STUDY ID Rpt MRI Cost STUDY ID Rpt MRI Cost Cost Cost Cost through Cost Cost Cost Testing Cost through testing testing

IEE11001 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE11001 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 0 IEE11002 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE11002 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 0 IEE11003 $4,299.29 $2,931.75 $2,100.00 $0.00 0 IEE11003 $4,299.29 $2,931.75 $2,100.00 $0.00 $2,200.00 0 IEE11006 $4,299.29 $2,931.75 $0.00 $0.00 1 IEE11006 $4,299.29 $2,931.75 $0.00 $0.00 $0.00 1 IEE11009 $4,299.29 $2,931.75 $2,100.00 $0.00 0 IEE11009 $4,299.29 $2,931.75 $2,100.00 $0.00 $2,200.00 0 IEE11010 $4,299.29 $2,931.75 $2,100.00 $0.00 0 IEE11010 $4,299.29 $2,931.75 $2,100.00 $0.00 $2,200.00 0 IEE11013 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE11013 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 1 IEE11015 $4,299.29 $2,931.75 $0.00 $0.00 1 IEE11015 $4,299.29 $2,931.75 $0.00 $0.00 $0.00 1 IEE11016 $4,299.29 $0.00 $0.00 $0.00 1 IEE11016 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE11020 $0.00 $0.00 $0.00 $0.00 0 IEE11020 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE11022 $4,299.29 $2,931.75 $2,100.00 $0.00 1 IEE11022 $4,299.29 $2,931.75 $2,100.00 $0.00 $0.00 1 IEE11026 $4,299.29 $0.00 $0.00 $0.00 1 IEE11026 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE11027 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE11027 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 0 IEE11028 $0.00 $0.00 $0.00 $0.00 0 IEE11028 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE11031 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE11031 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 0 IEE11033 $4,299.29 $0.00 $0.00 $0.00 1 IEE11033 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE11035 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE11035 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 0 IEE11038 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE11038 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 0 IEE11041 $0.00 $0.00 $0.00 $0.00 0 IEE11041 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12030 $0.00 $0.00 $0.00 $0.00 0 IEE12030 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12001 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE12001 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 0 IEE12002 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE12002 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 1 IEE12003 $4,299.29 $2,931.75 $2,100.00 $0.00 0 IEE12003 $4,299.29 $2,931.75 $2,100.00 $0.00 $2,200.00 0 IEE12004 $4,299.29 $0.00 $0.00 $0.00 1 IEE12004 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12005 $0.00 $0.00 $0.00 $0.00 0 IEE12005 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12006 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE12006 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 0 IEE12007 $4,299.29 $2,931.75 $2,100.00 $0.00 1 IEE12007 $4,299.29 $2,931.75 $2,100.00 $0.00 $0.00 1 IEE12008 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE12008 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 1 IEE12009 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE12009 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 0 IEE12010 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE12010 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 1 IEE12011 $4,299.29 $2,931.75 $2,100.00 $0.00 1 IEE12011 $4,299.29 $2,931.75 $2,100.00 $0.00 $0.00 1 IEE12012 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE12012 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 0 IEE12013 $4,299.29 $2,931.75 $2,100.00 $0.00 0 IEE12013 $4,299.29 $2,931.75 $2,100.00 $0.00 $2,200.00 0 IEE12014 $0.00 $0.00 $0.00 $0.00 0 IEE12014 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12015 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE12015 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 0 IEE12016 $4,299.29 $2,931.75 $2,100.00 $0.00 1 IEE12016 $4,299.29 $2,931.75 $2,100.00 $0.00 $0.00 1 IEE12017 $4,299.29 $0.00 $0.00 $0.00 1 IEE12017 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12019 $0.00 $0.00 $0.00 $0.00 0 IEE12019 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12020 $4,299.29 $0.00 $0.00 $0.00 1 IEE12020 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12021 $0.00 $0.00 $0.00 $0.00 0 IEE12021 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12022 $4,299.29 $0.00 $0.00 $0.00 1 IEE12022 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12023 $4,299.29 $0.00 $0.00 $0.00 1 IEE12023 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12024 $4,299.29 $0.00 $0.00 $0.00 1 IEE12024 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12025 $4,299.29 $2,931.75 $2,100.00 $0.00 0 IEE12025 $4,299.29 $2,931.75 $2,100.00 $0.00 $2,200.00 0 IEE12027 $0.00 $0.00 $0.00 $0.00 0 IEE12027 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12028 $4,299.29 $2,931.75 $2,100.00 $0.00 1 IEE12028 $4,299.29 $2,931.75 $2,100.00 $0.00 $0.00 1 IEE12029 $0.00 $0.00 $0.00 $0.00 0 IEE12029 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12031 $4,299.29 $0.00 $0.00 $0.00 1 IEE12031 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12032 $4,299.29 $0.00 $0.00 $0.00 1 IEE12032 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12033 $0.00 $0.00 $0.00 $0.00 0 IEE12033 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12036 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE12036 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 0 IEE12037 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE12037 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 1 IEE12038 $4,299.29 $0.00 $0.00 $0.00 1 IEE12038 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12039 $4,299.29 $0.00 $0.00 $0.00 1 IEE12039 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12040 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE12040 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 1 IEE12043 $4,299.29 $0.00 $0.00 $0.00 1 IEE12043 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE13014 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE13014 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 0 IEE13028 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE13028 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 1 IEE13038 $0.00 $0.00 $0.00 $0.00 0 IEE13038 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12045 $0.00 $0.00 $0.00 $0.00 0 IEE12045 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13001 $4,299.29 $0.00 $0.00 $0.00 1 IEE13001 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE13002 $4,299.29 $2,931.75 $0.00 $0.00 1 IEE13002 $4,299.29 $2,931.75 $0.00 $0.00 $0.00 1 IEE13003 $4,299.29 $2,931.75 $2,100.00 $0.00 0 IEE13003 $4,299.29 $2,931.75 $2,100.00 $0.00 $2,200.00 0 IEE13004 $4,299.29 $2,931.75 $2,100.00 $7,125.95 1 IEE13004 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $0.00 1 IEE13005 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE13005 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 0 IEE13006 $4,299.29 $2,931.75 $2,100.00 $0.00 0 IEE13006 $4,299.29 $2,931.75 $2,100.00 $0.00 $2,200.00 0 IEE13007 $4,299.29 $2,931.75 $2,100.00 $0.00 0 IEE13007 $4,299.29 $2,931.75 $2,100.00 $0.00 $2,200.00 0 IEE13008 $0.00 $0.00 $0.00 $0.00 0 IEE13008 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13009 $4,299.29 $2,931.75 $2,100.00 $0.00 0 IEE13009 $4,299.29 $2,931.75 $2,100.00 $0.00 $2,200.00 1 IEE13010 $4,299.29 $2,931.75 $2,100.00 $0.00 0 IEE13010 $4,299.29 $2,931.75 $2,100.00 $0.00 $2,200.00 0 IEE13011 $0.00 $0.00 $0.00 $0.00 0 IEE13011 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13012 $0.00 $0.00 $0.00 $0.00 0 IEE13012 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13013 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE13013 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 1 IEE13015 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE13015 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 0 IEE13020 $4,299.29 $0.00 $0.00 $0.00 1 IEE13020 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE13023 $0.00 $0.00 $0.00 $0.00 0 IEE13023 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13025 $0.00 $0.00 $0.00 $0.00 0 IEE13025 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13026 $0.00 $0.00 $0.00 $0.00 0 IEE13026 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13027 $4,299.29 $2,931.75 $2,100.00 $0.00 0 IEE13027 $4,299.29 $2,931.75 $2,100.00 $0.00 $2,200.00 0 IEE13034 $4,299.29 $0.00 $0.00 $0.00 1 IEE13034 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE13035 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE13035 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 1 IEE13036 $4,299.29 $2,931.75 $2,100.00 $0.00 0 IEE13036 $4,299.29 $2,931.75 $2,100.00 $0.00 $2,200.00 0 IEE13037 $0.00 $0.00 $0.00 $0.00 0 IEE13037 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13039 $4,299.29 $2,931.75 $2,100.00 $0.00 0 IEE13039 $4,299.29 $2,931.75 $2,100.00 $0.00 $2,200.00 0 IEE13041 $4,299.29 $2,931.75 $2,100.00 $0.00 1 IEE13041 $4,299.29 $2,931.75 $2,100.00 $0.00 $0.00 1 IEE13043 $4,299.29 $0.00 $0.00 $0.00 1 IEE13043 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE13044 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE13044 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 0 IEE13045 $4,299.29 $0.00 $0.00 $0.00 1 IEE13045 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE13046 $0.00 $0.00 $0.00 $0.00 0 IEE13046 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13047 $4,299.29 $2,931.75 $2,100.00 $0.00 0 IEE13047 $4,299.29 $2,931.75 $2,100.00 $0.00 $2,200.00 0 IEE13048 $4,299.29 $2,931.75 $2,100.00 $0.00 0 IEE13048 $4,299.29 $2,931.75 $2,100.00 $0.00 $2,200.00 0 IEE14023 $4,299.29 $2,931.75 $2,100.00 $0.00 0 IEE14023 $4,299.29 $2,931.75 $2,100.00 $0.00 $2,200.00 0 IEE13049 $4,299.29 $0.00 $0.00 $0.00 1 IEE13049 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE14001 $4,299.29 $0.00 $0.00 $0.00 1 IEE14001 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE14002 $0.00 $0.00 $0.00 $0.00 0 IEE14002 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14003 $4,299.29 $2,931.75 $2,100.00 $0.00 0 IEE14003 $4,299.29 $2,931.75 $2,100.00 $0.00 $2,200.00 0 IEE14004 $4,299.29 $0.00 $0.00 $0.00 1 IEE14004 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE14006 $4,299.29 $2,931.75 $2,100.00 $0.00 1 IEE14006 $4,299.29 $2,931.75 $2,100.00 $0.00 $0.00 1 IEE14010 $0.00 $0.00 $0.00 $0.00 0 IEE14010 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14012 $4,299.29 $2,931.75 $2,100.00 $0.00 1 IEE14012 $4,299.29 $2,931.75 $2,100.00 $0.00 $0.00 1 IEE14013 $0.00 $0.00 $0.00 $0.00 0 IEE14013 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14020 $4,299.29 $0.00 $0.00 $0.00 1 IEE14020 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE14026 $0.00 $0.00 $0.00 $0.00 0 IEE14026 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14027 $4,299.29 $0.00 $0.00 $0.00 1 IEE14027 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE14030 $4,299.29 $0.00 $0.00 $0.00 1 IEE14030 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE14033 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE14033 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 0 IEE14038 $4,299.29 $2,931.75 $2,100.00 $7,125.95 0 IEE14038 $4,299.29 $2,931.75 $2,100.00 $7,125.95 $2,200.00 0 IEE14046 $0.00 $0.00 $0.00 $0.00 0 IEE14046 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14050 $4,299.29 $0.00 $0.00 $0.00 1 IEE14050 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE14051 $0.00 $0.00 $0.00 $0.00 0 IEE14051 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14015 $4,299.29 $2,931.75 $2,100.00 $0.00 0 IEE14015 $4,299.29 $2,931.75 $2,100.00 $0.00 $2,200.00 0 IEE14053 $0.00 $0.00 $0.00 $0.00 0 IEE14053 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14054 $4,299.29 $2,931.75 $2,100.00 $0.00 0 IEE14054 $4,299.29 $2,931.75 $2,100.00 $0.00 $2,200.00 0 IEE15009 $4,299.29 $0.00 $0.00 $0.00 1 IEE15009 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 Scenario 3 (multigene pos 4) Scenario 4 (multigene pos 3)

Diagnosis Diagnosis Tier 1 Testing Tier 2 Testing Multigene Tier 3 Testing made Tier 1 Testing Tier 2 Testing Multigene Tier 3 Testing made STUDY ID Rpt MRI Cost STUDY ID Rpt MRI Cost Cost Cost Testing Cost Cost through Cost Cost Testing Cost Cost through testing testing

IEE11001 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $7,125.95 0 IEE11001 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $7,125.95 0 IEE11002 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $7,125.95 0 IEE11002 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $7,125.95 0 IEE11003 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 0 IEE11003 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 0 IEE11006 $4,299.29 $2,931.75 $0.00 $0.00 $0.00 1 IEE11006 $4,299.29 $2,931.75 $0.00 $0.00 $0.00 1 IEE11009 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 0 IEE11009 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 0 IEE11010 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 0 IEE11010 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 0 IEE11013 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 1 IEE11013 $4,299.29 $2,931.75 $2,200.00 $0.00 $0.00 1 IEE11015 $4,299.29 $2,931.75 $0.00 $0.00 $0.00 1 IEE11015 $4,299.29 $2,931.75 $0.00 $0.00 $0.00 1 IEE11016 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE11016 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE11020 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE11020 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE11022 $4,299.29 $2,931.75 $2,100.00 $0.00 $0.00 1 IEE11022 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 1 IEE11026 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE11026 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE11027 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $7,125.95 0 IEE11027 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $7,125.95 0 IEE11028 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE11028 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE11031 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $7,125.95 0 IEE11031 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $7,125.95 0 IEE11033 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE11033 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE11035 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $7,125.95 0 IEE11035 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $7,125.95 0 IEE11038 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $7,125.95 0 IEE11038 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $7,125.95 0 IEE11041 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE11041 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12030 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12030 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12001 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $7,125.95 0 IEE12001 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $7,125.95 0 IEE12002 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 1 IEE12002 $4,299.29 $2,931.75 $2,200.00 $0.00 $0.00 1 IEE12003 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 0 IEE12003 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 0 IEE12004 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12004 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12005 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12005 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12006 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $7,125.95 0 IEE12006 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $7,125.95 0 IEE12007 $4,299.29 $2,931.75 $2,100.00 $0.00 $0.00 1 IEE12007 $4,299.29 $2,931.75 $2,200.00 $0.00 $0.00 1 IEE12008 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 1 IEE12008 $4,299.29 $2,931.75 $2,200.00 $0.00 $0.00 1 IEE12009 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $7,125.95 0 IEE12009 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $7,125.95 0 IEE12010 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 1 IEE12010 $4,299.29 $2,931.75 $2,200.00 $0.00 $0.00 1 IEE12011 $4,299.29 $2,931.75 $2,100.00 $0.00 $0.00 1 IEE12011 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 1 IEE12012 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $7,125.95 0 IEE12012 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $7,125.95 0 IEE12013 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 0 IEE12013 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 0 IEE12014 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12014 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12015 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $7,125.95 0 IEE12015 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $7,125.95 0 IEE12016 $4,299.29 $2,931.75 $2,100.00 $0.00 $0.00 1 IEE12016 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 1 IEE12017 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12017 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12019 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12019 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12020 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12020 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12021 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12021 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12022 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12022 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12023 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12023 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12024 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12024 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12025 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 0 IEE12025 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 0 IEE12027 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12027 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12028 $4,299.29 $2,931.75 $2,100.00 $0.00 $0.00 1 IEE12028 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 1 IEE12029 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12029 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12031 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12031 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12032 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12032 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12033 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12033 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12036 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $7,125.95 0 IEE12036 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $7,125.95 0 IEE12037 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 1 IEE12037 $4,299.29 $2,931.75 $2,200.00 $0.00 $0.00 1 IEE12038 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12038 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12039 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12039 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12040 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 1 IEE12040 $4,299.29 $2,931.75 $2,200.00 $0.00 $0.00 1 IEE12043 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12043 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE13014 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $7,125.95 0 IEE13014 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $7,125.95 0 IEE13028 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 1 IEE13028 $4,299.29 $2,931.75 $2,200.00 $0.00 $0.00 1 IEE13038 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13038 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12045 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12045 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13001 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE13001 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE13002 $4,299.29 $2,931.75 $0.00 $0.00 $0.00 1 IEE13002 $4,299.29 $2,931.75 $0.00 $0.00 $0.00 1 IEE13003 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 0 IEE13003 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 0 IEE13004 $4,299.29 $2,931.75 $2,100.00 $0.00 $0.00 1 IEE13004 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $7,125.95 1 IEE13005 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $7,125.95 0 IEE13005 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $7,125.95 0 IEE13006 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 0 IEE13006 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 0 IEE13007 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 0 IEE13007 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 0 IEE13008 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13008 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13009 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 1 IEE13009 $4,299.29 $2,931.75 $2,200.00 $0.00 $0.00 1 IEE13010 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 0 IEE13010 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 0 IEE13011 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13011 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13012 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13012 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13013 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 1 IEE13013 $4,299.29 $2,931.75 $2,200.00 $0.00 $0.00 1 IEE13015 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $7,125.95 0 IEE13015 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $7,125.95 0 IEE13020 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE13020 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE13023 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13023 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13025 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13025 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13026 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13026 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13027 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 0 IEE13027 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 0 IEE13034 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE13034 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE13035 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 1 IEE13035 $4,299.29 $2,931.75 $2,200.00 $0.00 $0.00 1 IEE13036 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 0 IEE13036 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 0 IEE13037 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13037 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13039 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 0 IEE13039 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 0 IEE13041 $4,299.29 $2,931.75 $2,100.00 $0.00 $0.00 1 IEE13041 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 1 IEE13043 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE13043 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE13044 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $7,125.95 0 IEE13044 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $7,125.95 0 IEE13045 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE13045 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE13046 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13046 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13047 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 0 IEE13047 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 0 IEE13048 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 0 IEE13048 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 0 IEE14023 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 0 IEE14023 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 0 IEE13049 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE13049 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE14001 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE14001 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE14002 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14002 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14003 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 0 IEE14003 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 0 IEE14004 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE14004 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE14006 $4,299.29 $2,931.75 $2,100.00 $0.00 $0.00 1 IEE14006 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 1 IEE14010 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14010 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14012 $4,299.29 $2,931.75 $2,100.00 $0.00 $0.00 1 IEE14012 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 1 IEE14013 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14013 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14020 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE14020 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE14026 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14026 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14027 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE14027 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE14030 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE14030 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE14033 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $7,125.95 0 IEE14033 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $7,125.95 0 IEE14038 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $7,125.95 0 IEE14038 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $7,125.95 0 IEE14046 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14046 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14050 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE14050 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE14051 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14051 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14015 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 0 IEE14015 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 0 IEE14053 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14053 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14054 $4,299.29 $2,931.75 $2,100.00 $2,200.00 $0.00 0 IEE14054 $4,299.29 $2,931.75 $2,200.00 $2,100.00 $0.00 0 IEE15009 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE15009 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 Scenario 5 (multigene pos 2) Scenario 7 (multigene pos 2, no tier 2 or 3)

Diagnosis Diagnosis Tier 1 Testing Multigene Tier 2 Testing Tier 3 Testing made Tier 1 Testing DX Made Tier Multigene DX Made DX Made Rpt made STUDY ID Rpt MRI Cost STUDY ID Rpt MRI Cost Cost Testing Cost Cost Cost through Cost 1 Testing Cost Multigene MRI through testing testing

IEE11001 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $7,125.95 0 IEE11001 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE11002 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $7,125.95 0 IEE11002 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE11003 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 0 IEE11003 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE11006 $4,299.29 $2,200.00 $0.00 $0.00 $0.00 1 IEE11006 $4,299.29 0 $2,200.00 1 $0.00 0 1 IEE11009 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 0 IEE11009 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE11010 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 0 IEE11010 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE11013 $4,299.29 $2,200.00 $0.00 $0.00 $0.00 1 IEE11013 $4,299.29 0 $2,200.00 1 $0.00 0 1 IEE11015 $4,299.29 $2,200.00 $2,931.75 $0.00 $0.00 1 IEE11015 $4,299.29 0 $2,200.00 0 $0.00 0 0 IEE11016 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE11016 $4,299.29 1 $0.00 0 $0.00 0 1 IEE11020 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE11020 $0.00 0 $0.00 0 $0.00 0 0 IEE11022 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 1 IEE11022 $4,299.29 0 $2,200.00 0 $2,100.00 1 1 IEE11026 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE11026 $4,299.29 1 $0.00 0 $0.00 0 1 IEE11027 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $7,125.95 0 IEE11027 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE11028 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE11028 $0.00 0 $0.00 0 $0.00 0 0 IEE11031 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $7,125.95 0 IEE11031 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE11033 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE11033 $4,299.29 1 $0.00 0 $0.00 0 1 IEE11035 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $7,125.95 0 IEE11035 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE11038 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $7,125.95 0 IEE11038 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE11041 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE11041 $0.00 0 $0.00 0 $0.00 0 0 IEE12030 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12030 $0.00 0 $0.00 0 $0.00 0 0 IEE12001 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $7,125.95 0 IEE12001 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE12002 $4,299.29 $2,200.00 $0.00 $0.00 $0.00 1 IEE12002 $4,299.29 0 $2,200.00 1 $0.00 0 1 IEE12003 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 0 IEE12003 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE12004 $4,299.29 $2,200.00 $0.00 $0.00 $0.00 1 IEE12004 $4,299.29 0 $2,200.00 1 $0.00 0 1 IEE12005 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12005 $0.00 0 $0.00 0 $0.00 0 0 IEE12006 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $7,125.95 0 IEE12006 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE12007 $4,299.29 $2,200.00 $0.00 $0.00 $0.00 1 IEE12007 $4,299.29 0 $2,200.00 1 $0.00 0 1 IEE12008 $4,299.29 $2,200.00 $0.00 $0.00 $0.00 1 IEE12008 $4,299.29 0 $2,200.00 1 $0.00 0 1 IEE12009 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $7,125.95 0 IEE12009 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE12010 $4,299.29 $2,200.00 $0.00 $0.00 $0.00 1 IEE12010 $4,299.29 0 $2,200.00 1 $0.00 0 1 IEE12011 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 1 IEE12011 $4,299.29 0 $2,200.00 0 $2,100.00 1 1 IEE12012 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $7,125.95 0 IEE12012 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE12013 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 0 IEE12013 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE12014 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12014 $0.00 0 $0.00 0 $0.00 0 0 IEE12015 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $7,125.95 0 IEE12015 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE12016 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 1 IEE12016 $4,299.29 0 $2,200.00 0 $2,100.00 1 1 IEE12017 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12017 $4,299.29 1 $0.00 0 $0.00 0 1 IEE12019 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12019 $0.00 0 $0.00 0 $0.00 0 0 IEE12020 $4,299.29 $2,200.00 $0.00 $0.00 $0.00 1 IEE12020 $4,299.29 0 $2,200.00 1 $0.00 0 1 IEE12021 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12021 $0.00 0 $0.00 0 $0.00 0 0 IEE12022 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12022 $4,299.29 1 $0.00 0 $0.00 0 1 IEE12023 $4,299.29 $2,200.00 $0.00 $0.00 $0.00 1 IEE12023 $4,299.29 0 $2,200.00 1 $0.00 0 1 IEE12024 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12024 $4,299.29 1 $0.00 0 $0.00 0 1 IEE12025 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 0 IEE12025 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE12027 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12027 $0.00 0 $0.00 0 $0.00 0 0 IEE12028 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 1 IEE12028 $4,299.29 0 $2,200.00 0 $2,100.00 1 1 IEE12029 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12029 $0.00 0 $0.00 0 $0.00 0 0 IEE12031 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12031 $4,299.29 1 $0.00 0 $0.00 0 1 IEE12032 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12032 $4,299.29 1 $0.00 0 $0.00 0 1 IEE12033 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12033 $0.00 0 $0.00 0 $0.00 0 0 IEE12036 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $7,125.95 0 IEE12036 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE12037 $4,299.29 $2,200.00 $0.00 $0.00 $0.00 1 IEE12037 $4,299.29 0 $2,200.00 1 $0.00 0 1 IEE12038 $4,299.29 $2,200.00 $0.00 $0.00 $0.00 1 IEE12038 $4,299.29 0 $2,200.00 1 $0.00 0 1 IEE12039 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12039 $4,299.29 1 $0.00 0 $0.00 0 1 IEE12040 $4,299.29 $2,200.00 $0.00 $0.00 $0.00 1 IEE12040 $4,299.29 0 $2,200.00 1 $0.00 0 1 IEE12043 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE12043 $4,299.29 1 $0.00 0 $0.00 0 1 IEE13014 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $7,125.95 0 IEE13014 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE13028 $4,299.29 $2,200.00 $0.00 $0.00 $0.00 1 IEE13028 $4,299.29 0 $2,200.00 1 $0.00 0 1 IEE13038 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13038 $0.00 0 $0.00 0 $0.00 0 0 IEE12045 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE12045 $0.00 0 $0.00 0 $0.00 0 0 IEE13001 $4,299.29 $2,200.00 $0.00 $0.00 $0.00 1 IEE13001 $4,299.29 0 $2,200.00 1 $0.00 0 1 IEE13002 $4,299.29 $2,200.00 $2,931.75 $0.00 $0.00 1 IEE13002 $4,299.29 0 $2,200.00 0 $0.00 0 0 IEE13003 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 0 IEE13003 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE13004 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $7,125.95 1 IEE13004 $4,299.29 0 $2,200.00 0 $2,100.00 1 1 IEE13005 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $7,125.95 0 IEE13005 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE13006 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 0 IEE13006 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE13007 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 0 IEE13007 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE13008 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13008 $0.00 0 $0.00 0 $0.00 0 0 IEE13009 $4,299.29 $2,200.00 $0.00 $0.00 $0.00 1 IEE13009 $4,299.29 0 $2,200.00 1 $0.00 0 1 IEE13010 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 0 IEE13010 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE13011 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13011 $0.00 0 $0.00 0 $0.00 0 0 IEE13012 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13012 $0.00 0 $0.00 0 $0.00 0 0 IEE13013 $4,299.29 $2,200.00 $0.00 $0.00 $0.00 1 IEE13013 $4,299.29 0 $2,200.00 1 $0.00 0 1 IEE13015 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $7,125.95 0 IEE13015 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE13020 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE13020 $4,299.29 1 $0.00 0 $0.00 0 1 IEE13023 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13023 $0.00 0 $0.00 0 $0.00 0 0 IEE13025 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13025 $0.00 0 $0.00 0 $0.00 0 0 IEE13026 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13026 $0.00 0 $0.00 0 $0.00 0 0 IEE13027 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 0 IEE13027 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE13034 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE13034 $4,299.29 1 $0.00 0 $0.00 0 1 IEE13035 $4,299.29 $2,200.00 $0.00 $0.00 $0.00 1 IEE13035 $4,299.29 0 $2,200.00 1 $0.00 0 1 IEE13036 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 0 IEE13036 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE13037 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13037 $0.00 0 $0.00 0 $0.00 0 0 IEE13039 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 0 IEE13039 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE13041 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 1 IEE13041 $4,299.29 0 $2,200.00 0 $2,100.00 1 1 IEE13043 $4,299.29 $2,200.00 $0.00 $0.00 $0.00 1 IEE13043 $4,299.29 0 $2,200.00 1 $0.00 0 1 IEE13044 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $7,125.95 0 IEE13044 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE13045 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE13045 $4,299.29 1 $0.00 0 $0.00 0 1 IEE13046 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE13046 $0.00 0 $0.00 0 $0.00 0 0 IEE13047 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 0 IEE13047 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE13048 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 0 IEE13048 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE14023 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 0 IEE14023 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE13049 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE13049 $4,299.29 1 $0.00 0 $0.00 0 1 IEE14001 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE14001 $4,299.29 1 $0.00 0 $0.00 0 1 IEE14002 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14002 $0.00 0 $0.00 0 $0.00 0 0 IEE14003 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 0 IEE14003 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE14004 $4,299.29 $2,200.00 $0.00 $0.00 $0.00 1 IEE14004 $4,299.29 0 $2,200.00 1 $0.00 0 1 IEE14006 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 1 IEE14006 $4,299.29 0 $2,200.00 0 $2,100.00 1 1 IEE14010 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14010 $0.00 0 $0.00 0 $0.00 0 0 IEE14012 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 1 IEE14012 $4,299.29 0 $2,200.00 0 $2,100.00 1 1 IEE14013 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14013 $0.00 0 $0.00 0 $0.00 0 0 IEE14020 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE14020 $4,299.29 1 $0.00 0 $0.00 0 1 IEE14026 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14026 $0.00 0 $0.00 0 $0.00 0 0 IEE14027 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE14027 $4,299.29 1 $0.00 0 $0.00 0 1 IEE14030 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE14030 $4,299.29 1 $0.00 0 $0.00 0 1 IEE14033 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $7,125.95 0 IEE14033 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE14038 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $7,125.95 0 IEE14038 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE14046 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14046 $0.00 0 $0.00 0 $0.00 0 0 IEE14050 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE14050 $4,299.29 1 $0.00 0 $0.00 0 1 IEE14051 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14051 $0.00 0 $0.00 0 $0.00 0 0 IEE14015 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 0 IEE14015 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE14053 $0.00 $0.00 $0.00 $0.00 $0.00 0 IEE14053 $0.00 0 $0.00 0 $0.00 0 0 IEE14054 $4,299.29 $2,200.00 $2,931.75 $2,100.00 $0.00 0 IEE14054 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 IEE15009 $4,299.29 $0.00 $0.00 $0.00 $0.00 1 IEE15009 $4,299.29 1 $0.00 0 $0.00 0 1 Scenario 6 (multigene pos 2, Tier 2 after repeat MRI)

Diagnosis Diagnosis Tier 1 Testing DX Made Tier Multigene DX Made DX Made Rpt made DX Made Tier made STUDY ID Rpt MRI Cost Tier 2 Cost Cost 1 Testing Cost Multigene MRI through 2 through testing todate testing

IEE11001 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE11002 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE11003 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE11006 $4,299.29 0 $2,200.00 1 $0.00 0 1 $0.00 0 1 IEE11009 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE11010 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE11013 $4,299.29 0 $2,200.00 1 $0.00 0 1 $0.00 0 1 IEE11015 $4,299.29 0 $2,200.00 0 $0.00 0 0 $2,931.75 1 1 IEE11016 $4,299.29 1 $0.00 0 $0.00 0 1 $0.00 0 1 IEE11020 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE11022 $4,299.29 0 $2,200.00 0 $2,100.00 1 1 $0.00 0 1 IEE11026 $4,299.29 1 $0.00 0 $0.00 0 1 $0.00 0 1 IEE11027 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE11028 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE11031 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE11033 $4,299.29 1 $0.00 0 $0.00 0 1 $0.00 0 1 IEE11035 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE11038 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE11041 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE12030 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE12001 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE12002 $4,299.29 0 $2,200.00 1 $0.00 0 1 $0.00 0 1 IEE12003 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE12004 $4,299.29 0 $2,200.00 1 $0.00 0 1 $0.00 0 1 IEE12005 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE12006 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE12007 $4,299.29 0 $2,200.00 1 $0.00 0 1 $0.00 0 1 IEE12008 $4,299.29 0 $2,200.00 1 $0.00 0 1 $0.00 0 1 IEE12009 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE12010 $4,299.29 0 $2,200.00 1 $0.00 0 1 $0.00 0 1 IEE12011 $4,299.29 0 $2,200.00 0 $2,100.00 1 1 $0.00 0 1 IEE12012 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE12013 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE12014 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE12015 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE12016 $4,299.29 0 $2,200.00 0 $2,100.00 1 1 $0.00 0 1 IEE12017 $4,299.29 1 $0.00 0 $0.00 0 1 $0.00 0 1 IEE12019 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE12020 $4,299.29 0 $2,200.00 1 $0.00 0 1 $0.00 0 1 IEE12021 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE12022 $4,299.29 1 $0.00 0 $0.00 0 1 $0.00 0 1 IEE12023 $4,299.29 0 $2,200.00 1 $0.00 0 1 $0.00 0 1 IEE12024 $4,299.29 1 $0.00 0 $0.00 0 1 $0.00 0 1 IEE12025 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE12027 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE12028 $4,299.29 0 $2,200.00 0 $2,100.00 1 1 $0.00 0 1 IEE12029 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE12031 $4,299.29 1 $0.00 0 $0.00 0 1 $0.00 0 1 IEE12032 $4,299.29 1 $0.00 0 $0.00 0 1 $0.00 0 1 IEE12033 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE12036 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE12037 $4,299.29 0 $2,200.00 1 $0.00 0 1 $0.00 0 1 IEE12038 $4,299.29 0 $2,200.00 1 $0.00 0 1 $0.00 0 1 IEE12039 $4,299.29 1 $0.00 0 $0.00 0 1 $0.00 0 1 IEE12040 $4,299.29 0 $2,200.00 1 $0.00 0 1 $0.00 0 1 IEE12043 $4,299.29 1 $0.00 0 $0.00 0 1 $0.00 0 1 IEE13014 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE13028 $4,299.29 0 $2,200.00 1 $0.00 0 1 $0.00 0 1 IEE13038 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE12045 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE13001 $4,299.29 0 $2,200.00 1 $0.00 0 1 $0.00 0 1 IEE13002 $4,299.29 0 $2,200.00 0 $0.00 0 0 $2,931.75 1 1 IEE13003 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE13004 $4,299.29 0 $2,200.00 0 $2,100.00 1 1 $0.00 0 1 IEE13005 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE13006 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE13007 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE13008 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE13009 $4,299.29 0 $2,200.00 1 $0.00 0 1 $0.00 0 1 IEE13010 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE13011 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE13012 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE13013 $4,299.29 0 $2,200.00 1 $0.00 0 1 $0.00 0 1 IEE13015 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE13020 $4,299.29 1 $0.00 0 $0.00 0 1 $0.00 0 1 IEE13023 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE13025 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE13026 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE13027 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE13034 $4,299.29 1 $0.00 0 $0.00 0 1 $0.00 0 1 IEE13035 $4,299.29 0 $2,200.00 1 $0.00 0 1 $0.00 0 1 IEE13036 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE13037 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE13039 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE13041 $4,299.29 0 $2,200.00 0 $2,100.00 1 1 $0.00 0 1 IEE13043 $4,299.29 0 $2,200.00 1 $0.00 0 1 $0.00 0 1 IEE13044 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE13045 $4,299.29 1 $0.00 0 $0.00 0 1 $0.00 0 1 IEE13046 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE13047 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE13048 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE14023 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE13049 $4,299.29 1 $0.00 0 $0.00 0 1 $0.00 0 1 IEE14001 $4,299.29 1 $0.00 0 $0.00 0 1 $0.00 0 1 IEE14002 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE14003 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE14004 $4,299.29 0 $2,200.00 1 $0.00 0 1 $0.00 0 1 IEE14006 $4,299.29 0 $2,200.00 0 $2,100.00 1 1 $0.00 0 1 IEE14010 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE14012 $4,299.29 0 $2,200.00 0 $2,100.00 1 1 $0.00 0 1 IEE14013 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE14020 $4,299.29 1 $0.00 0 $0.00 0 1 $0.00 0 1 IEE14026 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE14027 $4,299.29 1 $0.00 0 $0.00 0 1 $0.00 0 1 IEE14030 $4,299.29 1 $0.00 0 $0.00 0 1 $0.00 0 1 IEE14033 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE14038 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE14046 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE14050 $4,299.29 1 $0.00 0 $0.00 0 1 $0.00 0 1 IEE14051 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE14015 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE14053 $0.00 0 $0.00 0 $0.00 0 0 $0.00 0 0 IEE14054 $4,299.29 0 $2,200.00 0 $2,100.00 0 0 $2,931.75 0 0 IEE15009 $4,299.29 1 $0.00 0 $0.00 0 1 $0.00 0 1 SCENARIOS IN WHICH ONLY INFANTS WITH ONGOING SEIZURES CONTINUE THROUGH DIAGNOSTIC PATHWAY Scenario 1-b (no multigene)

Diagnosis Tier 1 Testing Sz ongoing Tier 2 Testing Sz ongoing Sz ongoing Tier 3 Testing made STUDY ID Dx made yet? Dx made yet? Rpt MRI Cost Dx made yet? Dx made yet? Total Cost Cost >1m Cost >3m >6m Cost through testing

IEE11001 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 0 $16,456.99 IEE11002 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 0 $16,456.99 IEE11003 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE11006 $4,299.29 1 0 $2,931.75 1 1 $0.00 1 1 $0.00 1 1 $7,231.04 IEE11009 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 0 0 $0.00 0 0 $9,331.04 IEE11010 $4,299.29 1 0 $2,931.75 0 0 $0.00 0 0 $0.00 0 0 $7,231.04 IEE11013 $4,299.29 1 0 $2,931.75 0 0 $0.00 0 0 $0.00 0 0 $7,231.04 IEE11015 $4,299.29 1 0 $2,931.75 1 1 $0.00 1 1 $0.00 1 1 $7,231.04 IEE11016 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE11020 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE11022 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 1 $0.00 1 1 $9,331.04 IEE11026 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE11027 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 0 $16,456.99 IEE11028 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE11031 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 0 $16,456.99 IEE11033 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE11035 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 0 $16,456.99 IEE11038 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 0 $16,456.99 IEE11041 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE12030 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE12001 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 0 $16,456.99 IEE12002 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 0 $16,456.99 IEE12003 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE12004 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE12005 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE12006 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 0 $16,456.99 IEE12007 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 1 $0.00 1 1 $9,331.04 IEE12008 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 0 $16,456.99 IEE12009 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 0 $16,456.99 IEE12010 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 0 $16,456.99 IEE12011 $4,299.29 0 0 $0.00 0 0 $0.00 0 1 $0.00 1 1 $4,299.29 IEE12012 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 0 $16,456.99 IEE12013 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE12014 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE12015 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 0 $16,456.99 IEE12016 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 1 $0.00 1 1 $9,331.04 IEE12017 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE12019 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE12020 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE12021 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE12022 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE12023 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE12024 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE12025 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE12027 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE12028 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 1 $0.00 1 1 $9,331.04 IEE12029 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE12031 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE12032 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE12033 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE12036 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 0 $16,456.99 IEE12037 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 0 $16,456.99 IEE12038 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE12039 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE12040 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 0 $16,456.99 IEE12043 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE13014 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 0 $16,456.99 IEE13028 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 0 0 $0.00 0 0 $9,331.04 IEE13038 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE12045 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE13001 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE13002 $4,299.29 1 0 $2,931.75 1 1 $0.00 1 1 $0.00 1 1 $7,231.04 IEE13003 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE13004 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 1 $0.00 1 1 $9,331.04 IEE13005 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 0 $16,456.99 IEE13006 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE13007 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 0 0 $0.00 0 0 $9,331.04 IEE13008 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE13009 $4,299.29 1 0 $2,931.75 0 0 $0.00 0 0 $0.00 0 0 $7,231.04 IEE13010 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE13011 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE13012 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE13013 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 0 $16,456.99 IEE13015 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 0 $16,456.99 IEE13020 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE13023 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE13025 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE13026 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE13027 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE13034 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE13035 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE13036 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE13037 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE13039 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $0.00 0 0 $9,331.04 IEE13041 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 1 $0.00 1 1 $9,331.04 IEE13043 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE13044 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 0 $16,456.99 IEE13045 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE13046 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE13047 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $0.00 0 0 $9,331.04 IEE13048 $4,299.29 1 0 $2,931.75 0 0 $0.00 0 0 $0.00 0 0 $7,231.04 IEE14023 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE13049 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE14001 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE14002 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE14003 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 0 0 $0.00 0 0 $9,331.04 IEE14004 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE14006 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 1 $0.00 1 1 $9,331.04 IEE14010 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE14012 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 1 $0.00 1 1 $9,331.04 IEE14013 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE14020 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE14026 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE14027 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE14030 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE14033 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 0 $16,456.99 IEE14038 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 0 $16,456.99 IEE14046 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE14050 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE14051 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE14015 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE14053 $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 N/A N/A $0.00 IEE14054 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $0.00 0 0 $9,331.04 IEE15009 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 Scenario 2-b (multigene pos 5)

Diagnosis Tier 1 Testing Sz ongoing Tier 2 Testing Sz ongoing Sz ongoing Tier 3 Testing Multigene made STUDY ID Dx made yet? Dx made yet? Rpt MRI Cost Dx made yet? Dx made yet? Dx made yet? Total Cost Cost >1m Cost >3m >6m Cost Testing Cost through testing

IEE11001 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 $2,200.00 0 0 $18,656.99 IEE11002 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 $2,200.00 0 0 $18,656.99 IEE11003 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE11006 $4,299.29 1 0 $2,931.75 1 1 $0.00 1 1 $0.00 1 $0.00 1 1 $7,231.04 IEE11009 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 0 0 $0.00 0 $0.00 0 0 $9,331.04 IEE11010 $4,299.29 1 0 $2,931.75 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $7,231.04 IEE11013 $4,299.29 1 0 $2,931.75 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $7,231.04 IEE11015 $4,299.29 1 0 $2,931.75 1 1 $0.00 1 1 $0.00 1 $0.00 1 1 $7,231.04 IEE11016 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE11020 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE11022 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 1 $0.00 1 $0.00 1 1 $9,331.04 IEE11026 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE11027 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 $2,200.00 0 0 $18,656.99 IEE11028 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE11031 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 $2,200.00 0 0 $18,656.99 IEE11033 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE11035 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 $2,200.00 0 0 $18,656.99 IEE11038 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 $2,200.00 0 0 $18,656.99 IEE11041 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12030 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12001 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 $2,200.00 0 0 $18,656.99 IEE12002 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 $2,200.00 1 1 $18,656.99 IEE12003 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE12004 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12005 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12006 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 $2,200.00 0 0 $18,656.99 IEE12007 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 1 $0.00 1 $0.00 1 1 $9,331.04 IEE12008 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 $2,200.00 1 1 $18,656.99 IEE12009 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 $2,200.00 0 0 $18,656.99 IEE12010 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 $2,200.00 1 1 $18,656.99 IEE12011 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE12012 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 $2,200.00 0 0 $18,656.99 IEE12013 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE12014 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12015 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 $2,200.00 0 0 $18,656.99 IEE12016 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 1 $0.00 1 $0.00 1 1 $9,331.04 IEE12017 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12019 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12020 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12021 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12022 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12023 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12024 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12025 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE12027 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12028 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 1 $0.00 1 $0.00 1 1 $9,331.04 IEE12029 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12031 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12032 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12033 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12036 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 $2,200.00 0 0 $18,656.99 IEE12037 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 $2,200.00 1 1 $18,656.99 IEE12038 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12039 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12040 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 $2,200.00 1 1 $18,656.99 IEE12043 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE13014 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 $2,200.00 0 0 $18,656.99 IEE13028 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 0 0 $0.00 0 $0.00 0 0 $9,331.04 IEE13038 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12045 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13001 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE13002 $4,299.29 1 0 $2,931.75 1 1 $0.00 1 1 $0.00 1 $0.00 1 1 $7,231.04 IEE13003 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13004 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 1 $0.00 1 $0.00 1 1 $9,331.04 IEE13005 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 $2,200.00 0 0 $18,656.99 IEE13006 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13007 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 0 0 $0.00 0 $0.00 0 0 $9,331.04 IEE13008 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13009 $4,299.29 1 0 $2,931.75 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $7,231.04 IEE13010 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13011 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13012 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13013 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 $2,200.00 1 1 $18,656.99 IEE13015 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 $2,200.00 0 0 $18,656.99 IEE13020 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE13023 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13025 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13026 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13027 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13034 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE13035 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13036 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13037 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13039 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $0.00 0 $2,200.00 0 0 $11,531.04 IEE13041 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 1 $0.00 1 $0.00 1 1 $9,331.04 IEE13043 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE13044 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 $2,200.00 0 0 $18,656.99 IEE13045 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE13046 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13047 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $0.00 0 $2,200.00 0 0 $11,531.04 IEE13048 $4,299.29 1 0 $2,931.75 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $7,231.04 IEE14023 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13049 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14001 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14002 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14003 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 0 0 $0.00 0 $0.00 0 0 $9,331.04 IEE14004 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14006 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 1 $0.00 1 $0.00 1 1 $9,331.04 IEE14010 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14012 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 1 $0.00 1 $0.00 1 1 $9,331.04 IEE14013 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14020 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14026 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14027 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14030 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14033 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 $2,200.00 0 0 $18,656.99 IEE14038 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $7,125.95 0 $2,200.00 0 0 $18,656.99 IEE14046 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14050 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14051 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14015 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE14053 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14054 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $0.00 0 $2,200.00 0 0 $11,531.04 IEE15009 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 Scenario 3-b (multigene pos 4)

Diagnosis Tier 1 Testing Sz ongoing Tier 2 Testing Sz ongoing Sz ongoing Multigene Tier 3 Testing made STUDY ID Dx made yet? Dx made yet? Rpt MRI Cost Dx made yet? Dx made yet? Dx made yet? Total Cost Cost >1m Cost >3m >6m Testing Cost Cost through testing

IEE11001 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 0 $7,125.95 0 0 $18,656.99 IEE11002 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 0 $7,125.95 0 0 $18,656.99 IEE11003 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE11006 $4,299.29 1 0 $2,931.75 1 1 $0.00 1 1 $0.00 1 $0.00 1 1 $7,231.04 IEE11009 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 0 0 $0.00 0 $0.00 0 0 $9,331.04 IEE11010 $4,299.29 1 0 $2,931.75 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $7,231.04 IEE11013 $4,299.29 1 0 $2,931.75 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $7,231.04 IEE11015 $4,299.29 1 0 $2,931.75 1 1 $0.00 1 1 $0.00 1 $0.00 1 1 $7,231.04 IEE11016 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE11020 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE11022 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 1 $0.00 1 $0.00 1 1 $9,331.04 IEE11026 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE11027 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 0 $7,125.95 0 0 $18,656.99 IEE11028 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE11031 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 0 $7,125.95 0 0 $18,656.99 IEE11033 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE11035 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 0 $7,125.95 0 0 $18,656.99 IEE11038 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 0 $7,125.95 0 0 $18,656.99 IEE11041 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12030 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12001 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 0 $7,125.95 0 0 $18,656.99 IEE12002 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 1 $0.00 1 1 $11,531.04 IEE12003 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE12004 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12005 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12006 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 0 $7,125.95 0 0 $18,656.99 IEE12007 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 1 $0.00 1 $0.00 1 1 $9,331.04 IEE12008 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 1 $0.00 1 1 $11,531.04 IEE12009 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 0 $7,125.95 0 0 $18,656.99 IEE12010 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 1 $0.00 1 1 $11,531.04 IEE12011 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE12012 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 0 $7,125.95 0 0 $18,656.99 IEE12013 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE12014 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12015 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 0 $7,125.95 0 0 $18,656.99 IEE12016 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 1 $0.00 1 $0.00 1 1 $9,331.04 IEE12017 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12019 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12020 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12021 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12022 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12023 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12024 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12025 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE12027 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12028 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 1 $0.00 1 $0.00 1 1 $9,331.04 IEE12029 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12031 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12032 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12033 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12036 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 0 $7,125.95 0 0 $18,656.99 IEE12037 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 1 $0.00 1 1 $11,531.04 IEE12038 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12039 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12040 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 1 $0.00 1 1 $11,531.04 IEE12043 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE13014 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 0 $7,125.95 0 0 $18,656.99 IEE13028 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 0 0 $0.00 0 $0.00 0 0 $9,331.04 IEE13038 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12045 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13001 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE13002 $4,299.29 1 0 $2,931.75 1 1 $0.00 1 1 $0.00 1 $0.00 1 1 $7,231.04 IEE13003 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13004 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 1 $0.00 1 $0.00 1 1 $9,331.04 IEE13005 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 0 $7,125.95 0 0 $18,656.99 IEE13006 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13007 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 0 0 $0.00 0 $0.00 0 0 $9,331.04 IEE13008 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13009 $4,299.29 1 0 $2,931.75 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $7,231.04 IEE13010 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13011 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13012 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13013 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 1 $0.00 1 1 $11,531.04 IEE13015 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 0 $7,125.95 0 0 $18,656.99 IEE13020 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE13023 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13025 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13026 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13027 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13034 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE13035 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13036 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13037 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13039 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 0 $0.00 0 0 $11,531.04 IEE13041 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 1 $0.00 1 $0.00 1 1 $9,331.04 IEE13043 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE13044 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 0 $7,125.95 0 0 $18,656.99 IEE13045 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE13046 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13047 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 0 $0.00 0 0 $11,531.04 IEE13048 $4,299.29 1 0 $2,931.75 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $7,231.04 IEE14023 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13049 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14001 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14002 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14003 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 0 0 $0.00 0 $0.00 0 0 $9,331.04 IEE14004 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14006 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 1 $0.00 1 $0.00 1 1 $9,331.04 IEE14010 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14012 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 1 $0.00 1 $0.00 1 1 $9,331.04 IEE14013 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14020 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14026 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14027 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14030 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14033 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 0 $7,125.95 0 0 $18,656.99 IEE14038 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 0 $7,125.95 0 0 $18,656.99 IEE14046 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14050 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14051 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14015 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE14053 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14054 $4,299.29 1 0 $2,931.75 1 0 $2,100.00 1 0 $2,200.00 0 $0.00 0 0 $11,531.04 IEE15009 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 Scenario 4-b (multigene pos 3)

Diagnosis Tier 1 Testing Sz ongoing Tier 2 Testing Sz ongoing Multigene Sz ongoing Repeat MRI Tier 3 Testing made STUDY ID Dx made yet? Dx made yet? Dx made yet? Dx made yet? Dx made yet? Total Cost Cost >1m Cost >3m Testing Cost >6m Cost Cost through testing

IEE11001 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE11002 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE11003 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE11006 $4,299.29 1 0 $2,931.75 1 1 $0.00 1 1 $0.00 1 $0.00 1 1 $7,231.04 IEE11009 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 0 0 $0.00 0 $0.00 0 0 $9,431.04 IEE11010 $4,299.29 1 0 $2,931.75 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $7,231.04 IEE11013 $4,299.29 1 0 $2,931.75 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $7,231.04 IEE11015 $4,299.29 1 0 $2,931.75 1 1 $0.00 1 1 $0.00 1 $0.00 1 1 $7,231.04 IEE11016 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE11020 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE11022 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 1 $0.00 1 1 $11,531.04 IEE11026 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE11027 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE11028 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE11031 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE11033 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE11035 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE11038 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE11041 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12030 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12001 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE12002 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 1 $0.00 1 $0.00 1 1 $9,431.04 IEE12003 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE12004 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12005 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12006 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE12007 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 1 $0.00 1 $0.00 1 1 $9,431.04 IEE12008 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 1 $0.00 1 $0.00 1 1 $9,431.04 IEE12009 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE12010 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 1 $0.00 1 $0.00 1 1 $9,431.04 IEE12011 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE12012 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE12013 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE12014 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12015 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE12016 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 1 $0.00 1 1 $11,531.04 IEE12017 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12019 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12020 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12021 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12022 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12023 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12024 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12025 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE12027 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12028 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 1 $0.00 1 1 $11,531.04 IEE12029 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12031 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12032 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12033 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12036 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE12037 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 1 $0.00 1 $0.00 1 1 $9,431.04 IEE12038 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12039 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12040 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 1 $0.00 1 $0.00 1 1 $9,431.04 IEE12043 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE13014 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE13028 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 0 1 $0.00 1 $0.00 1 1 $9,431.04 IEE13038 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12045 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13001 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE13002 $4,299.29 1 0 $2,931.75 1 1 $0.00 1 1 $0.00 1 $0.00 1 1 $7,231.04 IEE13003 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13004 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 1 $0.00 1 1 $11,531.04 IEE13005 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE13006 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13007 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 0 0 $0.00 0 $0.00 0 0 $9,431.04 IEE13008 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13009 $4,299.29 1 0 $2,931.75 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $7,231.04 IEE13010 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13011 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13012 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13013 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 1 $0.00 1 $0.00 1 1 $9,431.04 IEE13015 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE13020 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE13023 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13025 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13026 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13027 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13034 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE13035 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13036 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13037 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13039 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 0 $0.00 0 0 $11,531.04 IEE13041 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 1 $0.00 1 1 $11,531.04 IEE13043 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE13044 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE13045 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE13046 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13047 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 0 $0.00 0 0 $11,531.04 IEE13048 $4,299.29 1 0 $2,931.75 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $7,231.04 IEE14023 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13049 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14001 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14002 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14003 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 0 0 $0.00 0 $0.00 0 0 $9,431.04 IEE14004 $4,299.29 1 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14006 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 1 $0.00 1 1 $11,531.04 IEE14010 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14012 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 1 $0.00 1 1 $11,531.04 IEE14013 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14020 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14026 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14027 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14030 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14033 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE14038 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE14046 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14050 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14051 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14015 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE14053 $0.00 N/A N/A $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14054 $4,299.29 1 0 $2,931.75 1 0 $2,200.00 1 0 $2,100.00 0 $0.00 0 0 $11,531.04 IEE15009 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 Scenario 5-b (multigene pos 2)

Diagnosis Tier 1 Testing Sz ongoing Multigene Sz ongoing Tier 2 Testing Sz ongoing Repeat MRI Tier 3 Testing made STUDY ID Dx made yet? Dx made yet? Dx made yet? Dx made yet? Dx made yet? Total Cost Cost >1m Testing Cost >3m Cost >6m Cost Cost through testing

IEE11001 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE11002 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE11003 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE11006 $4,299.29 1 0 $2,200.00 1 1 $0.00 1 1 $0.00 1 $0.00 1 1 $6,499.29 IEE11009 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 0 0 $0.00 0 $0.00 0 0 $9,431.04 IEE11010 $4,299.29 1 0 $2,200.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $6,499.29 IEE11013 $4,299.29 1 0 $2,200.00 0 1 $0.00 0 1 $0.00 1 $0.00 1 1 $6,499.29 IEE11015 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 1 $0.00 1 $0.00 1 1 $9,431.04 IEE11016 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE11020 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE11022 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 1 $0.00 1 1 $11,531.04 IEE11026 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE11027 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE11028 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE11031 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE11033 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE11035 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE11038 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE11041 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12030 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12001 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE12002 $4,299.29 1 0 $2,200.00 1 1 $0.00 1 1 $0.00 1 $0.00 1 1 $6,499.29 IEE12003 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE12004 $4,299.29 1 0 $2,200.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $6,499.29 IEE12005 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12006 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE12007 $4,299.29 1 0 $2,200.00 1 1 $0.00 1 1 $0.00 1 $0.00 1 1 $6,499.29 IEE12008 $4,299.29 1 0 $2,200.00 1 1 $0.00 1 1 $0.00 1 $0.00 1 1 $6,499.29 IEE12009 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE12010 $4,299.29 1 0 $2,200.00 1 1 $0.00 1 1 $0.00 1 $0.00 1 1 $6,499.29 IEE12011 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE12012 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE12013 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE12014 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12015 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE12016 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 1 $0.00 1 1 $11,531.04 IEE12017 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12019 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12020 $4,299.29 1 0 $2,200.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $6,499.29 IEE12021 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12022 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12023 $4,299.29 1 0 $2,200.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $6,499.29 IEE12024 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12025 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE12027 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12028 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 1 $0.00 1 1 $11,531.04 IEE12029 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12031 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12032 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12033 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12036 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE12037 $4,299.29 1 0 $2,200.00 1 1 $0.00 1 1 $0.00 1 $0.00 1 1 $6,499.29 IEE12038 $4,299.29 1 0 $2,200.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $6,499.29 IEE12039 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE12040 $4,299.29 1 0 $2,200.00 1 1 $0.00 1 1 $0.00 1 $0.00 1 1 $6,499.29 IEE12043 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE13014 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE13028 $4,299.29 1 0 $2,200.00 1 1 $0.00 0 1 $0.00 1 $0.00 1 1 $6,499.29 IEE13038 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE12045 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13001 $4,299.29 1 0 $2,200.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $6,499.29 IEE13002 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 1 $0.00 1 $0.00 1 1 $9,431.04 IEE13003 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13004 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 1 $0.00 1 1 $11,531.04 IEE13005 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE13006 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13007 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 0 0 $0.00 0 $0.00 0 0 $9,431.04 IEE13008 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13009 $4,299.29 1 0 $2,200.00 0 1 $0.00 0 1 $0.00 1 $0.00 1 1 $6,499.29 IEE13010 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13011 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13012 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13013 $4,299.29 1 0 $2,200.00 1 1 $0.00 1 1 $0.00 1 $0.00 1 1 $6,499.29 IEE13015 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE13020 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE13023 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13025 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13026 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13027 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13034 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE13035 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13036 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13037 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13039 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 0 $0.00 0 0 $11,531.04 IEE13041 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 1 $0.00 1 1 $11,531.04 IEE13043 $4,299.29 1 0 $2,200.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $6,499.29 IEE13044 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE13045 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE13046 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE13047 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 0 $0.00 0 0 $11,531.04 IEE13048 $4,299.29 1 0 $2,200.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $6,499.29 IEE14023 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE13049 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14001 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14002 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14003 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 0 0 $0.00 0 $0.00 0 0 $9,431.04 IEE14004 $4,299.29 1 0 $2,200.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $6,499.29 IEE14006 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 1 $0.00 1 1 $11,531.04 IEE14010 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14012 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 1 $0.00 1 1 $11,531.04 IEE14013 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14020 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14026 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14027 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14030 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14033 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE14038 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 0 $7,125.95 0 0 $18,656.99 IEE14046 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14050 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 IEE14051 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14015 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 $0.00 0 0 $4,299.29 IEE14053 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 $0.00 0 0 $0.00 IEE14054 $4,299.29 1 0 $2,200.00 1 0 $2,931.75 1 0 $2,100.00 0 $0.00 0 0 $11,531.04 IEE15009 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 1 $0.00 1 1 $4,299.29 Scenario 7-b (multigene pos 2, rpt MRI pos 3, nil else)

Diagnosis Tier 1 Testing Sz ongoing Multigene Sz ongoing made STUDY ID Dx made yet? Dx made yet? Rpt MRI cost Dx made yet? Total Cost Cost >1m Testing Cost >3m through testing

IEE11001 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE11002 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE11003 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE11006 $4,299.29 1 0 $2,200.00 1 1 $0.00 1 1 $6,499.29 IEE11009 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE11010 $4,299.29 1 0 $2,200.00 0 0 $0.00 0 0 $6,499.29 IEE11013 $4,299.29 1 0 $2,200.00 0 1 $0.00 1 1 $6,499.29 IEE11015 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE11016 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE11020 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE11022 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 1 $8,599.29 IEE11026 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE11027 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE11028 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE11031 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE11033 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE11035 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE11038 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE11041 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE12030 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE12001 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE12002 $4,299.29 1 0 $2,200.00 1 1 $0.00 1 1 $6,499.29 IEE12003 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE12004 $4,299.29 1 0 $2,200.00 N/A 1 $0.00 1 1 $6,499.29 IEE12005 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE12006 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE12007 $4,299.29 1 0 $2,200.00 1 1 $0.00 1 1 $6,499.29 IEE12008 $4,299.29 1 0 $2,200.00 1 1 $0.00 1 1 $6,499.29 IEE12009 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE12010 $4,299.29 1 0 $2,200.00 1 1 $0.00 1 1 $6,499.29 IEE12011 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE12012 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE12013 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE12014 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE12015 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE12016 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 1 $8,599.29 IEE12017 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE12019 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE12020 $4,299.29 1 0 $2,200.00 N/A 1 $0.00 1 1 $6,499.29 IEE12021 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE12022 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE12023 $4,299.29 1 0 $2,200.00 N/A 1 $0.00 1 1 $6,499.29 IEE12024 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE12025 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE12027 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE12028 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 1 $8,599.29 IEE12029 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE12031 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE12032 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE12033 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE12036 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE12037 $4,299.29 1 0 $2,200.00 1 1 $0.00 1 1 $6,499.29 IEE12038 $4,299.29 1 0 $2,200.00 N/A 1 $0.00 1 1 $6,499.29 IEE12039 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE12040 $4,299.29 1 0 $2,200.00 1 1 $0.00 1 1 $6,499.29 IEE12043 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE13014 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE13028 $4,299.29 1 0 $2,200.00 1 1 $0.00 1 1 $6,499.29 IEE13038 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE12045 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE13001 $4,299.29 1 0 $2,200.00 N/A 1 $0.00 1 1 $6,499.29 IEE13002 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE13003 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE13004 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 1 $8,599.29 IEE13005 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE13006 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE13007 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE13008 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE13009 $4,299.29 1 0 $2,200.00 0 1 $0.00 1 1 $6,499.29 IEE13010 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE13011 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE13012 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE13013 $4,299.29 1 0 $2,200.00 1 1 $0.00 1 1 $6,499.29 IEE13015 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE13020 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE13023 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE13025 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE13026 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE13027 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE13034 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE13035 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE13036 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE13037 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE13039 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE13041 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 1 $8,599.29 IEE13043 $4,299.29 1 0 $2,200.00 N/A 1 $0.00 1 1 $6,499.29 IEE13044 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE13045 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE13046 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE13047 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE13048 $4,299.29 1 0 $2,200.00 0 0 $0.00 0 0 $6,499.29 IEE14023 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE13049 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE14001 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE14002 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE14003 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE14004 $4,299.29 1 0 $2,200.00 N/A 1 $0.00 1 1 $6,499.29 IEE14006 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 1 $8,599.29 IEE14010 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE14012 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 1 $8,599.29 IEE14013 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE14020 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE14026 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE14027 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE14030 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE14033 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE14038 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE14046 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE14050 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 IEE14051 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE14015 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE14053 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE14054 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $8,599.29 IEE15009 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 1 1 $4,299.29 Scenario 6-b (multigene pos 2, rpt MRI pos 3, Tier 2)

Diagnosis Tier 1 Testing Sz ongoing Multigene Sz ongoing Sz ongoing DX made on made STUDY ID Dx made yet? Dx made yet? Rpt MRI cost Dx made yet? Tier 2 Cost Total Cost Cost >1m Testing Cost >3m >6m Tier 2? through testing

IEE11001 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 0 $2,931.75 0 0 $11,531.04 IEE11002 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 0 $2,931.75 0 0 $11,531.04 IEE11003 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE11006 $4,299.29 1 0 $2,200.00 1 1 $0.00 1 1 $0.00 0 1 $6,499.29 IEE11009 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $0.00 0 0 $8,599.29 IEE11010 $4,299.29 1 0 $2,200.00 0 0 $0.00 0 0 $0.00 0 0 $6,499.29 IEE11013 $4,299.29 1 0 $2,200.00 0 1 $0.00 0 1 $0.00 0 1 $6,499.29 IEE11015 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 0 $2,931.75 1 1 $11,531.04 IEE11016 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $4,299.29 IEE11020 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE11022 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 1 $0.00 0 1 $8,599.29 IEE11026 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $4,299.29 IEE11027 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 0 $2,931.75 0 0 $11,531.04 IEE11028 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE11031 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 0 $2,931.75 0 0 $11,531.04 IEE11033 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $4,299.29 IEE11035 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 0 $2,931.75 0 0 $11,531.04 IEE11038 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 0 $2,931.75 0 0 $11,531.04 IEE11041 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE12030 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE12001 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 0 $2,931.75 0 0 $11,531.04 IEE12002 $4,299.29 1 0 $2,200.00 1 1 $0.00 1 1 $0.00 0 1 $6,499.29 IEE12003 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE12004 $4,299.29 1 0 $2,200.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $6,499.29 IEE12005 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE12006 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 0 $2,931.75 0 0 $11,531.04 IEE12007 $4,299.29 1 0 $2,200.00 1 1 $0.00 1 1 $0.00 0 1 $6,499.29 IEE12008 $4,299.29 1 0 $2,200.00 1 1 $0.00 1 1 $0.00 0 1 $6,499.29 IEE12009 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 0 $2,931.75 0 0 $11,531.04 IEE12010 $4,299.29 1 0 $2,200.00 1 1 $0.00 1 1 $0.00 0 1 $6,499.29 IEE12011 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE12012 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 0 $2,931.75 0 0 $11,531.04 IEE12013 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE12014 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE12015 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 0 $2,931.75 0 0 $11,531.04 IEE12016 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 1 $0.00 0 1 $8,599.29 IEE12017 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $4,299.29 IEE12019 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE12020 $4,299.29 1 0 $2,200.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $6,499.29 IEE12021 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE12022 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $4,299.29 IEE12023 $4,299.29 1 0 $2,200.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $6,499.29 IEE12024 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $4,299.29 IEE12025 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE12027 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE12028 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 1 $0.00 0 1 $8,599.29 IEE12029 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE12031 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $4,299.29 IEE12032 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $4,299.29 IEE12033 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE12036 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 0 $2,931.75 0 0 $11,531.04 IEE12037 $4,299.29 1 0 $2,200.00 1 1 $0.00 1 1 $0.00 0 1 $6,499.29 IEE12038 $4,299.29 1 0 $2,200.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $6,499.29 IEE12039 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $4,299.29 IEE12040 $4,299.29 1 0 $2,200.00 1 1 $0.00 1 1 $0.00 0 1 $6,499.29 IEE12043 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $4,299.29 IEE13014 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 0 $2,931.75 0 0 $11,531.04 IEE13028 $4,299.29 1 0 $2,200.00 1 1 $0.00 0 1 $0.00 0 1 $6,499.29 IEE13038 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE12045 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE13001 $4,299.29 1 0 $2,200.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $6,499.29 IEE13002 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 0 $2,931.75 1 1 $11,531.04 IEE13003 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE13004 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 1 $0.00 0 1 $8,599.29 IEE13005 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 0 $2,931.75 0 0 $11,531.04 IEE13006 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE13007 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $0.00 0 0 $8,599.29 IEE13008 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE13009 $4,299.29 1 0 $2,200.00 0 1 $0.00 0 1 $0.00 0 1 $6,499.29 IEE13010 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE13011 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE13012 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE13013 $4,299.29 1 0 $2,200.00 1 1 $0.00 1 1 $0.00 0 1 $6,499.29 IEE13015 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 0 $2,931.75 0 0 $11,531.04 IEE13020 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $4,299.29 IEE13023 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE13025 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE13026 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE13027 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE13034 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $4,299.29 IEE13035 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE13036 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE13037 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE13039 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 0 $2,931.75 0 0 $11,531.04 IEE13041 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 1 $0.00 0 1 $8,599.29 IEE13043 $4,299.29 1 0 $2,200.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $6,499.29 IEE13044 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 0 $2,931.75 0 0 $11,531.04 IEE13045 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $4,299.29 IEE13046 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE13047 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 0 $2,931.75 0 0 $11,531.04 IEE13048 $4,299.29 1 0 $2,200.00 0 0 $0.00 0 0 $0.00 0 0 $6,499.29 IEE14023 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE13049 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $4,299.29 IEE14001 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $4,299.29 IEE14002 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE14003 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 0 0 $0.00 0 0 $8,599.29 IEE14004 $4,299.29 1 0 $2,200.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $6,499.29 IEE14006 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 1 $0.00 0 1 $8,599.29 IEE14010 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE14012 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 1 $0.00 0 1 $8,599.29 IEE14013 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE14020 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $4,299.29 IEE14026 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE14027 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $4,299.29 IEE14030 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $4,299.29 IEE14033 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 0 $2,931.75 0 0 $11,531.04 IEE14038 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 0 $2,931.75 0 0 $11,531.04 IEE14046 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE14050 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $4,299.29 IEE14051 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE14015 $4,299.29 0 0 $0.00 0 0 $0.00 0 0 $0.00 0 0 $4,299.29 IEE14053 $0.00 N/A 0 $0.00 N/A 0 $0.00 N/A 0 $0.00 0 0 $0.00 IEE14054 $4,299.29 1 0 $2,200.00 1 0 $2,100.00 1 0 $2,931.75 0 0 $11,531.04 IEE15009 $4,299.29 N/A 1 $0.00 N/A 1 $0.00 N/A 1 $0.00 0 1 $4,299.29 Confirmation Number of costs saved suspected through Total Cost of diagnoses # of tests Tier 1 cost Tier 2 cost Repeat MRI Tier 3 cost avoidance tiered testing needing Cost of Cost per Additional # of Cost per Cost per # of tests # of tests Tier # of tests # of tests savings savings cost savings savings (diagnosis Net Costs Additional Scenario Step 1 Step 2 Step 3 Step 4 Step 5 (without confirmation diagnostic TOTAL COST incremental Cost of Diagnoses Diagnosis patient Tier 1 Tier 2 Repeat Tier 3 Multigene through through through through confirmed avoided Total Cost confirmatory with tests not confirmation diagnosis Multigene MRI avoidance avoidance avoidance avoidance with WES costs) in the instead of diagnostic single gene pathway testing)

All infants investigated until aetiology identified or reach end of pathway Scenario 1 Tier 1 Tier 2 Repeat MRI Tier 3 $859,838.79 10 $27,547.62 $887,386.41 39 $22,753.50 $10,318.45 86 59 56 28 0 $0.00 $0.00 $0.00 $0.00 $0.00 $0.00 Scenario 2 Tier 1 Tier 2 Repeat MRI Tier 3 Multigene $963,238.79 10 $27,547.62 $990,786.41 49 $20,220.13 $10,340.00 $11,520.77 86 59 56 28 47 $0.00 $0.00 $0.00 $0.00 $103,400.00 $0.00 $0.00 $103,400.00 Scenario 3 Tier 1 Tier 2 Repeat MRI Multigene Tier 3 $891,979.29 10 $27,547.62 $919,526.91 49 $18,765.86 $3,214.05 $10,692.17 86 59 56 18 47 $0.00 $0.00 $0.00 -$71,259.50 $103,400.00 $0.00 $71,259.50 $32,140.50 Scenario 4 Tier 1 Tier 2 Multigene Repeat MRI Tier 3 $895,805.24 9 $26,446.62 $922,251.86 49 $18,821.47 $3,486.55 $10,723.86 86 59 45 19 56 $0.00 $0.00 -$23,100.00 -$64,133.55 $123,200.00 -$1,101.00 $88,334.55 $34,865.45 Scenario 5 Tier 1 Multigene Tier 2 Repeat MRI Tier 3 $882,624.24 2 $14,051.62 $896,675.86 49 $18,299.51 $928.94 $10,426.46 86 47 45 19 66 $0.00 -$35,181.00 -$23,100.00 -$64,133.55 $145,200.00 -$13,496.00 $135,910.55 $9,289.45 Scenario 7 Tier 1 Multigene Repeat MRI $609,438.94 0 $0.00 $609,438.94 47 $12,966.79 -$34,743.43 $7,086.50 86 0 45 0 66 $0.00 -$172,973.25 -$23,100.00 -$199,526.60 $145,200.00 -$27,547.62 $423,147.47 -$277,947.47 Scenario 6 Tier 1 Multigene Repeat MRI Tier 2 $723,777.19 2 $14,051.62 $737,828.81 49 $15,057.73 -$14,955.76 $8,579.40 86 39 45 0 66 $0.00 -$58,635.00 -$23,100.00 -$199,526.60 $145,200.00 -$13,496.00 $294,757.60 -$149,557.60

Infants continue through pathway only if seizures are ongoing until aetiology identified or reach end of pathway Scenario 1-b Tier 1 Tier 2 Repeat MRI Tier 3 $757,522.24 10 $27,547.62 $785,069.86 39 $20,130.00 $9,128.72 86 46 39 24 0 $0.00 $0.00 $0.00 $0.00 $0.00 $0.00 Scenario 2-b Tier 1 Tier 2 Repeat MRI Tier 3 Multigene $816,922.24 10 $27,547.62 $844,469.86 44 $19,192.50 $11,880.00 $9,819.42 86 46 39 24 27 $0.00 $0.00 $0.00 $0.00 $59,400.00 $0.00 $0.00 $59,400.00 Scenario 3-b Tier 1 Tier 2 Repeat MRI Multigene Tier 3 $774,166.54 10 $27,547.62 $801,714.16 44 $18,220.78 $3,328.86 $9,322.26 86 46 39 18 27 $0.00 $0.00 $0.00 -$42,755.70 $59,400.00 $0.00 $42,755.70 $16,644.30 Scenario 4-b Tier 1 Tier 2 Multigene Repeat MRI Tier 3 $777,466.54 9 $26,446.62 $803,913.16 45 $17,864.74 $3,140.55 $9,347.83 86 46 28 18 39 $0.00 $0.00 -$23,100.00 -$42,755.70 $85,800.00 -$1,101.00 $66,956.70 $18,843.30 Scenario 5-b Tier 1 Multigene Tier 2 Repeat MRI Tier 3 $770,153.79 2 $14,051.62 $784,205.41 47 $16,685.22 -$108.06 $9,118.67 86 33 28 18 53 $0.00 -$38,112.75 -$23,100.00 -$42,755.70 $116,600.00 -$13,496.00 $117,464.45 -$864.45 Scenario 7-b Tier 1 Multigene Repeat MRI $555,638.94 0 $0.00 $555,638.94 45 $12,347.53 -$38,238.49 $6,460.92 86 0 33 0 53 $0.00 -$134,860.50 -$12,600.00 -$171,022.80 $116,600.00 -$27,547.62 $346,030.92 -$229,430.92 Scenario 6-b Tier 1 Multigene Repeat MRI Tier 2 $623,069.19 2 $14,051.62 $637,120.81 47 $13,555.76 -$18,493.63 $7,408.38 86 23 33 0 53 $0.00 -$67,430.25 -$12,600.00 -$171,022.80 $116,600.00 -$13,496.00 $264,549.05 -$147,949.05

Minerva Access is the Institutional Repository of The University of Melbourne

Author/s: Howell, Katherine Brooke

Title: The epidemiology and aetiologies of the severe epilepsies of infancy

Date: 2016

Persistent Link: http://hdl.handle.net/11343/129828

File Description: The epidemiology and aetiologies of the severe epilepsies of infancy

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