Identification and Evaluation of Novel Epilepsy-Associated Variants Public
Butler, Kameryn (Spring 2018)
Published
Abstract
Epilepsy is a neurological disorder characterized by recurrent, unprovoked seizures caused by excessive neuronal synchrony and hyperexcitability. Although epilepsy can develop following insults such as head trauma, stroke, and infection, genetic factors are predicted to play a role in approximately 70-80% of epilepsy cases. To date, mutations in over 150 genes have been identified in different types of epilepsy; however, these mutations account for only a small fraction of the estimated genetic contribution to epilepsy. We collaborated with EGL Genetics, a CLIA-certified and CAP-accredited laboratory, to examine available sequence data from clinically-referred epilepsy patients. Upon referral, patient DNA samples were screened using the Epilepsy and Seizure Disorders (ESD) panel, a sequencing panel of 110 known epilepsy genes. The ESD panel is derived from a larger Mendeliome library of approximately 4,800 evidence-based disease genes, making this a valuable resource for the identification of putative disease-causing alleles as well as new disease associations. We determined the diagnostic yield of the ESD panel to be approximately 18% based on a review of panel results from 339 epilepsy patients, with most pathogenic variants affecting a small number of genes. From this analysis, we identified several factors that are likely to improve diagnostic yield. Additionally, we discovered five novel missense variants in the voltage-gated sodium channel gene SCN8A. Investigation of these variants revealed that both de novo and inherited SCN8A variants contribute to epilepsy, with inherited variants producing less severe forms of disease. Furthermore, we identified four regions of the sodium channel where disease variants cluster and discovered that an alternative coding exon of SCN8A was being overlooked by diagnostic laboratories. This dissertation also covers the examination of variants from the Mendeliome library from individuals with negative or inconclusive ESD panel results. Novel variants were identified in GABRA5 and GABRA2, two genes not previously associated with disease, from individuals with severe early-onset epilepsy and developmental delay. Functional evaluation of these GABAA receptor pore mutations revealed potential gain- and loss-of-function changes. Overall, these results highlight the usefulness of utilizing clinical sequencing data for research-based epilepsy gene and variant discovery.
Table of Contents
CHAPTER 1: EPILEPSY GENE AND VARIANT DISCOVERY ______________ 1
1.1 Overview __________________________________________________________ 2
1.2 General overview of the genetics of epilepsy _______________________________ 2
1.2.1 Types of epilepsy _____________________________________________ 3
1.2.2 The early genetics of epilepsy ___________________________________ 4
1.2.3 Epilepsy genetics in the 21st century ______________________________ 4
1.3 Genetic testing for epilepsy _____________________________________________ 5
1.3.1 Chromosomal microarrays and copy number variants ________________ 6
1.3.2 Next-generation sequencing ____________________________________ 7
1.3.2.1 Targeted gene panels ____________________________________ 8
1.3.2.2 Whole-exome sequencing ________________________________ 8
1.3.2.3 Whole-genome sequencing ______________________________ 10
1.3.3 De novo variants ____________________________________________ 11
1.3.4 The problem of VUS (and GUS) ________________________________ 11
1.3.5 Phenotype-first approach & Genetic heterogeneity __________________ 12
1.3.6 Genotype-first approach & Phenotypic heterogeneity ________________ 13
1.4 Using available sequencing data for epilepsy gene discovery __________________ 14
1.4.1 EGL generates sequence data from hundreds of epilepsy patients ______ 14
1.4.2 The Epilepsy and Seizure Disorders (ESD) Panel ___________________ 15
1.4.3 The ‘Mendeliome’ sequencing library ___________________________ 16
1.4.4 Advantages of using available data from clinical testing ______________ 17
1.4.5 Limitations of using available data from clinical testing ______________ 17
1.5 Project Aims ______________________________________________________ 18
CHAPTER 2: DIAGNOSTIC YIELD FROM 339 EPILEPSY PATIENTS SCREENED ON A CLINICAL GENE PANEL ____________________________ 19
2.1 Summary __________________________________________________________ 20
2.2 Introduction ________________________________________________________ 21
2.3 Methods and Materials ________________________________________________ 22
2.4 Results ____________________________________________________________ 24
2.4.1 Patient Demographics ________________________________________ 24
2.4.2 Yield from the Epilepsy and Seizure Disorders Panel ________________ 25
2.5 Discussion _________________________________________________________ 32
2.6 Acknowledgments ___________________________________________________ 34
CHAPTER 3: DE NOVO AND INHERITED SCN8A EPILEPSY MUTATIONS DETECTED BY GENE PANEL ANALYSIS ______________________________ 35
3.1 Summary _________________________________________________________ 36
3.2 Introduction _______________________________________________________ 37
3.3 Methods and Materials _______________________________________________ 40
3.4 Results ___________________________________________________________ 41
3.4.1 Identification of novel SCN8A epilepsy mutations __________________ 41
3.4.2 Patient Descriptions _________________________________________ 41
3.4.2.1 Patient 1: SCN8A, c.2287A>G, p.I763V ___________________ 41
3.4.2.2 Patient 2: SCN8A, c.3985A>G, p.N1329D __________________ 46
3.4.2.3 Patient 3: SCN8A, c.3995T>G, p.L1332R __________________ 47
3.4.2.4 Patient 4: SCN8A, c.5630A>G, p.N1877S __________________ 48
3.4.2.5 Patient 5: SCN8A, c.302A>G, p.K101R ____________________ 50
3.4.3 SCN8A variation in other neurodevelopmental disorders _____________ 52
3.4.4 Mutation clusters within the Nav1.6 channel _______________________ 54
3.5 Discussion _________________________________________________________ 55
3.6 Acknowledgments ___________________________________________________ 63
CHAPTER 4: DE NOVO VARIANTS IN GABRA2 AND GABRA5 ALTER RECEPTOR FUNCTION AND CONTRIBUTE TO EARLY-ONSET EPILEPSY _________________________________________________________________ 64
4.1 Summary _________________________________________________________ 65
4.2 Introduction _______________________________________________________ 66
4.3 Methods and Materials _______________________________________________ 67
4.4 Results ___________________________________________________________ 73
4.4.1 GABAA Receptor variants detected from individuals with epilepsy ____ 73
4.4.2 GABRA5 c.880G>C (p.V294L) ________________________________ 74
4.4.3 α5(V294L)β2γ2s Receptors are more sensitive to GABA but exhibit increased desensitization _____________________________________ 79
4.4.4 GABRA2 c.875C>A (p.T292K) ________________________________ 82
4.4.5 α2(T292K)β2γ2s Receptors are tonically open and unresponsive to GABA __________________________________________________________ 83
4.4.6 GABRB3 c.902C>T (p.P301L) _________________________________ 86
4.4.7 α1β3(P301L)γ2s Receptors are less sensitive to GABA and produce less GABA-evoked current _______________________________________ 87
4.5 Discussion ________________________________________________________ 89
4.6 Acknowledgments __________________________________________________ 97
CHAPTER 5: CONCLUSIONS AND FUTURE DIRECTIONS _______________ 98
5.1 Summary _________________________________________________________ 99
5.2 Improving gene panel analysis for epilepsy ______________________________ 99
5.3 What defines an “epilepsy” gene? ______________________________________ 102
5.4 The move towards WES and WGS _____________________________________ 103
5.5 Solving the missing genetics of epilepsy _________________________________ 104
5.5.1 Missed coding variants _____________________________________ 104
5.5.2 Noncoding variation _______________________________________ 105
5.5.3 Polygenic inheritance, Modifiers, and Dual diagnoses _____________ 106
5.6 Future directions ___________________________________________________ 108
REFERENCES _____________________________________________________ 112
APPENDIX A: IDENTIFICATION OF COMPOUND HETEROZYGOUS VARIANTS IN CACNA2D2 ____________________________________________ 135
APPENDIX B: POSITIVE FINDINGS FROM THE MENDELIOME _________ 142
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