UC Irvine UC Irvine Electronic Theses and Dissertations Title Exome Sequencing Reveals Gene Variants Involved in Methylation, Acetylation, and Chromatin Remodeling in Patients with Autism Permalink https://escholarship.org/uc/item/4br5n3vx Author Bevilacqua, Jennifer Ann Publication Date 2014 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, IRVINE Exome Sequencing Reveals Gene Variants Involved in Methylation, Acetylation, and Chromatin Remodeling in Patients with Autism THESIS submitted in partial satisfaction of the requirements for the degree of MASTER OF SCIENCE In Genetic Counseling By Jennifer Ann Bevilacqua Thesis Committee: Professor Moyra Smith, Chair Associate Adjunct Professor Pamela Flodman Assistant Clinical Professor Kathryn Singh 2014 © 2014 Jennifer Ann Bevilacqua DEDICATION To my parents and family, I could never thank you enough for your love, support, and encouragement to be all that I can be. You never doubted me and you always kept me moving in the right direction, especially when it seemed impossible to take that next step. I could never have made it this far without you. To all my teachers and mentors at UCI, Your guidance has and will continue to provide me with the skills I need to succeed in my genetic counseling career in the years to come. You helped me to grow into a confident, smart professional and I hope to make you all proud. To my classmates, I had no idea how important you would all become to me, but I am so glad that we were thrown together in this program! I could never have gotten through this without your support, encouragement, laughter, and friendship. You can do anything for two years and we did! Congratulations to all of you! ii TABLE OF CONTENTS Page LIST OF FIGURES iv LIST OF TABLES iv ACKNOWLEDGEMENTS v ABSTRACT OF THE THESIS vi INTRODUCTION 1 Overview of autism 2 Epigenetics 7 Overview of the folate cycle 11 Folate and neural tube defects 15 Gene and environmental interactions of the “epigenetic pool” 17 Exome sequencing and analysis 18 Novel variant classification – In silico prediction tools 20 Epistasis 22 METHODS 24 Subject recruitment 24 Exome sequencing 24 Variant filtering and classification 24 RESULTS 26 DISCUSSION 37 CONCLUSION 45 REFERENCES 47 APPENDIX 53 iii LIST OF FIGURES Page FIGURE 1 CNV at 2q37.3 including HDAC4 in patient with autism 5 FIGURE 2 CNV at 15q24.2 including SIN3A in patient with autism 6 FIGURE 3 The epigenome and chromatin organization 10 FIGURE 4 The folate and methionine cycle overview 12 FIGURE 5 The folate cycle 13 FIGURE 6 The methionine cycle 14 LIST OF TABLES Page TABLE 1 Exome variants in seven families with autism 27 TABLE 2 Rare and low frequency variants related to methylation, 31 acetylation, or chromatin remodeling in families and probands with autism TABLE 3 Common variants in the folate one-carbon pathway 34 iv ACKNOWLEDGEMENTS I would like to thank my committee chair, Professor Moyra Smith, and my committee members, Pamela Flodman and Kathryn Singh for their guidance and help in completing this manuscript. I would also like to thank our research participants for their time and interest in helping us to explore the causes of autism and attempt to uncover some of the mysteries of this condition. Their contributions are essential to our work and I hope research may someday provide them with the answers they and families like them continue to seek. I thank the Nature Publishing Group, Elsevier, and the American Society for Nutrition for their permission to include copyrighted figures as part of my thesis. v ABSTRACT OF THE THESIS Exome Sequencing Reveals Gene Variants Involved in Methylation, Acetylation, and Chromatin Remodeling in Patients with Autism By Jennifer Ann Bevilacqua Master of Science in Genetic Counseling University of California, Irvine, 2014 Professor Moyra Smith, Chair Autism is a complex and both genetically and phenotypically heterogeneous neurodevelopmental condition. Some clear genetic causes have been identified but for most cases of autism the cause is unknown. Previous studies attempting to fill in some of this gap in understanding have suggested a link between autism and variants in genes related to epigenetic control of gene expression including those involved in methylation and chromatin modification. We analyzed whole exome sequencing data for seven families and probands with autism for novel, rare, and common protein damaging variants and found 22 variants in 20 genes related to methylation, acetylation, and chromatin remodeling. Our analysis also revealed a burden of eight inherited variants in the one-carbon metabolism pathway in an autistic proband. These findings suggest that both defects in enzymes related to epigenetic regulation and the interactions of multiple related variants may each provide insight into the pathophysiology of autism. vi INTRODUCTION The traditional view of genetic disease is based in the central dogma of a DNA sequence transcribed into mRNA and further translated into protein. This doctrine puts a huge emphasis on the importance of the sequence of DNA nucleotides. Under this simplistic view, the sequence of bases has an exclusive impact on the final protein product and ultimately the function of the tissues and systems of the body. Advances in technology have focused on discovering the DNA sequence of protein coding genes with the goal of associating mutations with disease. Complex diseases, however, have proved difficult to explain using these methods. Sequence based methods have made it clear that complex human diseases like autism spectrum disorder are not easily explained by monogenic mutations. Other causes of autism including multifactorial influence and the epistatic effect of multiple genes have been investigated and focus has recently, focus turned to epigenetic modification as a possible explanation for complex neurological disorders. The goal of this study is to explore defects in enzymes involved in epigenetic control of gene function and chromatin modification as a possible underlying mechanism for autism. We also aim to explore the possibility of contributions of several interacting genes in a single individual to development of autism and whether variants with potential for autism causation might either arise de novo or be inherited from a parent. 1 Overview of autism Autism is a common but complex neurodevelopmental disorder characterized by deficiencies in communication, social interactions, and repetitive behaviors. Autism is a highly variable condition with symptoms ranging from complete lack of verbal communication and cognitive impairment, to characteristic repetitive behaviors, to mild social deficiency. It is because of this variability that there has recently been a shift in nomenclature to the term “autism spectrum disorder” which better reflects the wide range of phenotypes that encompass the condition [1]. ASD affects about 1 in 88 children in the United States according to Center for Disease Control estimates and the number of diagnoses is increasing over time [2]. This increase may be due to broadening diagnostic criteria, increased awareness, and the need for a formal diagnosis to gain access to appropriate therapies [3]. Due to the widespread prevalence of autism and this condition’s profound impact on affected families, research investigating the causes of autism is ongoing. Clearly identified monogenic disorders have been shown to cause autism. Fragile X syndrome affects 1/3,500-1/9,000 individuals and is associated with an unstable tri-nucleotide expansion in the FMR1 gene coding for the FMRP protein. The expansion of a CGG repeat results in abnormal methylation of the FMR1 gene and reduction in FMRP protein transcription leading to developmental delays, intellectual disabilities, and characteristic facial features. About 18-33% of patients with Fragile X syndrome meet diagnostic criteria for autism, and account for approximately 2% of all autistic individuals [4, 5]. Mutations in the TSC1 and TSC2 genes associated with tuberous sclerosis (TS) are also associated with an increased risk for autism. This autosomal dominant disorder is characterized by hamartomas, skin lesions, and epilepsy. As many as 30% of patients affected with TS have autism as a feature of the disorder [4]. Studies of samples 2 of autistic patients have found about 1% of patients have autism as a result of TS [5-7]. Another single gene disorder associated with autism is Rett syndrome, which is caused by mutations in MECP2. Clinical features of classic Rett syndrome include epilepsy, stereotypic hand wringing, breathing abnormalities, and developmental regression. Studies have found between 58-78% of children with Rett syndrome meet criteria for autism spectrum disorder [8, 9]. Other syndromic causes of autism include CHARGE syndrome, Down syndrome, and Angelman syndrome as well as other chromosomal and single gene disorders [5, 10]. More important than the incidence of autism in these syndromes may be the clues they provide for the underlying mechanism behind the development of autistic symptoms. Fragile X, TS, Rett syndrome, Down syndrome and other conditions affect behavior, communication, and intelligence in unique ways but all can result in autism. While autism can be considered a symptom of these conditions, understanding the connection between these genetic etiologies and effects on brain development could contribute to a more generalized explanation of non-syndromic neurological