Identification of Genetic and Epigenetic Factors regulating cell death and proliferation in cerebellum-related brain disorders Open Access

Kim, Hye Rim (Spring 2019)

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Molecular characterization is the key to understanding disease pathophysiology and developing effective therapeutic agents. However, incomplete (or reduced) penetrance and numerous genetic and epigenetic alterations irrelevant to disease progression mask the identification of bona fide disease-associated factors. Furthermore, genome-wide association studies are limited for the discovery of common variants associated with complex and common disorders, and therefore, novel approaches are needed to determine true pathological variants in rare and complex disorders. In addition, abnormal changes in the epigenome are deemed as key determinants in many diseases, but their role in pathogenesis remains to be understood.

We recently utilized a three-step gene discovery strategy to facilitate the identification of novel genetic factors implicated in amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease. Using whole-genome sequencing (WGS), we first identified genetic variants in 135 candidate genes associated with age-of-onset in patients with the G4C2 repeat expansion in the C9orf72 gene (step 1). We then performed an unbiased genetic screen using a Drosophila model expressing 30 repeats of G4C2, identifying 18 genetic factors modifying G4C2 repeat-associated toxicity (step 2). To further test the association of the 18 genes with sporadic ALS risk, gene-based statistical analyses of targeted resequencing and WGS identified rare variants in MYH15 as a modifying factor of ALS risk. We further demonstrated that MYH15 modulates the toxicity caused by poly-dipeptides produced from the expanded G4C2 repeat.

The cerebellum is critical for motor movements, and thus, neurogenesis in the cerebellum must be sophisticatedly orchestrated for normal neuronal activity. Epigenetic modifications play a critical role in postnatal and adult neurogenesis, but the role of 5-hydroxymethylcytosine (5hmC), an abundant epigenetic factor, in this process remains to be elucidated. We performed genome-wide 5hmC profiling to characterize the genomic loci enriched with 5hmC throughout the processes of neurodevelopment and aging. We further investigated the role of 5hmC alterations in Medulloblastoma (MB), a tumor of the cerebellum. Collectively, these studies highlight the effectiveness of our novel approach to facilitate the identification of genetic modifiers in rare and complex disorders and expand our understanding of epigenetic dynamics in the context of both normal development/aging and diseases.

Table of Contents


1.1. Author’s Contribution and Acknowledgement of Reproduction. 1

1.2. Challenges in rare variant association studies of complex neurological disorders 2

1.2.1. Family-based sequencing studies 3

1.2.2. Sampling of affected patients with extreme phenotypes. 3

1.2.3. Whole-Genome Sequencing. 4

1.2.4. Targeted sequencing of prioritized candidate genes. 5

1.2.5. Rare-variant association testing for sequencing data with the Sequence Kernel Association Test (SKAT) 6

1.2.6. Functional annotation of rare variants. 7

1.2.7. Functional genomics using a Drosophila model 8

1.3. The significance of epigenetics in cellular functions and diseases. 10

1.3.1. DNA modifications in genome: cytosine modifications and beyond. 11

1.3.2. Technologies for genome-wide DNA modifications. 14

1.3.3. Distinct genomic localization of TET proteins: intrinsic structural difference and the interaction with extrinsic factors 16

1.3.4. Significance of epigenetic alterations in pediatric cancer 17

1.4. Summary of background information and dissertation goals. 18

CHAPTER 2: Rare Variants in MYH15 Modify Amyotrophic Lateral Sclerosis Risk. 20

2.1. Author’s Contribution and Acknowledgement of Reproduction. 20

2.2. Introduction. 21

2.3. Materials and Methods. 23

2.4. Results. 29

2.5. Discussion. 39

2.6. Acknowledgements. 42

CHAPTER 3: Aging-related epigenetic dynamics in cerebellum.. 43

3.1. Introduction. 43

3.2. Materials and Methods. 45

3.3. Results. 48

3.4. Discussion. 60

CHAPTER 4: TET1-mediated 5-hydroxymethylcytosine Alteration in the pathogenesis of Medulloblastoma 62

4.1. Introduction. 62

4.2. Materials and Methods. 64

4.3. Results. 69

4.4. Discussion. 86

CHAPTER 5: Summary. 89

5.1. Summary of key findings. 89

5.2. Clinical implications. 92



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