Identification and Evaluation of Novel Epilepsy-Associated Variants Restricted; Files Only

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 21^st 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

 

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Genetics and Molecular Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Genetics
Palabra Clave	SCN8A Human Genetics Epilepsy GABA-A receptors Genetic testing
Committee Chair / Thesis Advisor	Escayg, Andrew, Emory University
Committee Members	Zwick, Michael, Emory University Caspary, Tamara, Emory University Hegde, Madhuri, Emory University Cubells, Joseph, Emory University

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