Towards a Better Understanding of the Genetic Contributions to Epilepsy Pubblico
Mattison, Kari (Spring 2022)
Abstract
Epilepsy, characterized by recurrent and unprovoked seizures, is a common neurological disorder affecting more than 3 million individuals in the United States. Use of next-generation sequencing in genetic testing of epilepsy patients has led to the identification of over 200 known and evidence-based epilepsy genes, representing diverse biological functions. The goal of this dissertation was to gain a better understanding of the genetic contributions to epilepsy. We utilized three approaches for the identification of candidate disease variants. The first approach was to analyze gene panel data consisting of approximately 4,800 evidence-based disease genes from 460 patients with epilepsy. We identified 8 variants in SLC6A1 from this data set which was a similar to the diagnostic yield of established epilepsy genes, SCN2A and CDKL5. We showed, for the first time, through functional assays that SLC6A1 variants identified in epilepsy patients result in decreased GABA re-uptake. In the second approach, we analyzed whole exome sequencing data from 218 patients with epilepsy. From this we identified a single de novo, heterozygous variant in ATP6V0C (p.A138P). Using GeneMatcher and other publicly available datasets, we subsequently identified 25 additional patients with ATP6V0C variants. To confirm pathogenicity of the identified variants, we developed functional assays using Saccharomyces cerevisiae which demonstrated that the ATP6V0C variants result in loss of vacuolar ATPase function. Our work resulted in the largest cohort to-date of patients with ATP6V0C variants and provides strong support for ATP6V0C as an epilepsy gene. Lastly, we performed whole genome sequencing in two brothers with epilepsy from a consanguineous family to identify variants within shared regions of homozygosity. We identified a homozygous variant, p.G228R, in CNTNAP2 and subsequently found co-segregation of the same variant in an unrelated family with overlapping clinical presentations. Taken together, three epilepsy genes were identified, each with a unique disease mechanism and function related to neuronal signaling. The knowledge gained from the identification and functional analysis of variants can provide insight into treatment options and/or development of precision therapies for patients.
Table of Contents
Chapter 1 – Introduction 1
1.1 Overview 2
1.2 Genetics of Epilepsy 3
1.2.1 Heritability 6
1.2.2 Modes of Inheritance 6
1.2.2.1 Autosomal Dominant 6
1.2.2.2 Autosomal Recessive 7
1.2.2.3 X-Linked 7
1.2.2.4 Other Factors 8
1.3 Diversifying out of the “Channelopathy” era 9
1.3.1 Enzymes 9
1.3.2 Transporters 10
1.3.3 Chromatin Remodelers and Nucleic Acid Binding Proteins 10
1.3.4 Other 11
1.4 Identifying Pathogenic Variants 11
1.4.1. Genetic Linkage and Targeted Sequencing 12
1.4.2 Copy Number Variants and Gene Panels 12
1.4.3 Whole Exome and Whole Genome Sequencing 13
1.5 Follow-up on VUS’s and GUS’s 13
1.5.1 Classification of Variants 14
1.5.2 Functional Testing 17
1.5.3 Problem of Singleton Cases 17
1.6 Benefits of a Genetic Diagnosis 18
1.6.1 Therapeutics 19
1.6.2 Support Groups 20
1.7 Summary and Goals of this Dissertation 20
Chapter 2 – SLC6A1 Variants Identified in Epilepsy Patients Reduce γ-Aminobutyric Acid Transport 22
2.1 Summary 23
2.2 Introduction 24
2.3 Subjects and Methods 24
2.4 Results 26
2.4.1 SLC6A1 Variants Identified in Individuals with Epilepsy 26
2.4.2 GABA Transport is Reduced by Variants Identified in Epilepsy Patients 29
2.4.3 c.850-2A>G Affects the Splicing of SLC6A1 30
2.5 Discussion 32
2.6 Acknowledgements 34
Chapter 3 – Variants in ATP6V0C Associated with Epilepsy Decrease the Functon of the Vacuoloar ATPase 35
3.1 Summary 36
3.2 Introduction 38
3.3 Materials and Methods 42
3.4 Results 47
3.4.1 Identification of ATP6V0C Variants in Patients 47
3.4.2 ATP6V0C Variants Cause a Human Syndrome of Developmental Delay, Epilepsy, and Intellectual Disability 50
3.4.3 ATP6V0C Knockdown in Drosophila Results in Seizure Phenotype 51
3.4.4 ATP6V0C Variants Are Predicted to Interfere with V-ATPase Rotary Mechanism 53
3.4.5 ATP6V0C Patient Variants are Deleterious in Yeast 55
3.4.6 Assessment of Three Patient Variants in C. elegans 62
3.5 Discussion 67
3.6 Acknowledgements and Funding 72
Chapter 4 – Novel Missense CNTNAP2 Variant Identified in Two Consanguineous Pakistani Families with Epilepsy and Intellectual Disability 73
4.1 Summary 74
4.2 Introduction 75
4.3 Subjects and Methods 76
4.4 Results 79
4.4.1 Clinical Presentation and Family History 79
4.4.1.1 Family 1 79
4.4.1.2 Family 2 82
4.4.2 Genomic Analyses 84
4.4.2.1 Family 1 84
4.4.2.2 Family 2 87
4.4.3 Relatedness and Linkage Analysis 87
4.5 Discussion 88
4.6 Acknowledgements 94
Chapter 5 – Discussion 95
5.1 Summary 96
5.2 Considerations in Genetic Testing and Variant Evaluation 99
5.2.1 Types of Tests Used 99
5.2.2 Burden for Pathogenicity 100
5.2.3 Ignoring the “Unknown” 100
5.3 How do we Find the “Missing” Variants? 101
5.3.1 Non-coding Variants 102
5.3.1.1 Untranslated Regions 102
5.3.1.2 DNA Regulatory Elements 103
5.3.1.3 non-coding RNAs 103
5.3.1.4 Therapeutic Strategies for Targeting Non-coding Variants 104
5.3.2 Alternative and Poison Exons 104
5.3.3 Mosaic Variants 105
5.4 Future Directions for ATP6V0C and Epilepsy 106
5.4.1 Mechanistic Studies 106
5.4.2 Therapeutic Strategies 109
5.4.2.1 Overexpression of wild-type ATP6V0C 109
5.4.2.2 Gene Editing 112
5.4.2.3 High Throughput Screening of AEDs and Novel Compounds 112
5.4.3 Longitudinal Studies 113
5.5 Future Directions for Genetic Testing 114
5.5.1 Gene-specific Databases 114
5.5.2 Reevaluation of data 115
5.5.3 Long-read WGS 115
5.6 Overall Conclusions 116
References 117
Appendix A – Supporting Data for Chapter 3 142
Appendix B – Results from Whole Genome Sequencing in Eight Consanguineous Families with Epilepsy 154
B.1 Summary 155
B.2 Methods 158
B.3 Results and Discussion 162
B.3.1 Family 201 162
B.3.2 Family 204 162
B.3.3 Family 205 164
B.3.4 Family 210 166
B.3.5 Family 220 168
B.3.6 Family 225 170
B.4 Conclusions and Future Directions 171
Appendix C – Individual Contributions to Chapter and Appendices 173
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