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Functional Genomics Studies of Human Brain Development and Implications for Autism Spectrum Disorder

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Functional Genomics Studies of Human Brain Development and Implications for Autism Spectrum Disorder Functional Genomics Studies of Human Brain Development and Implications for Autism Spectrum Disorder Mark Nicholas Ziats Robinson College University of Cambridge This dissertation is submitted for the degree of Doctor of Philosophy November 2013 For Mom, Dad, Ann, and Catherine 2 Declaration This dissertation is the result of my own work and contains nothing that is the outcome of work done in collaboration, except as specifically described in the Appendix. The length of this thesis does not exceed the 60,000 word limit and it has been typeset using the specifications set by the Biology Degree Committee. This dissertation is not similar to any other that I submitted for a degree, diploma, or other qualification at any other University. Furthermore, I state that no part of this dissertation has been, or is concurrently being, submitted for any degree, diploma, or other qualification. Mark N. Ziats November 2013 3 Summary Human neurodevelopment requires the coordinated expression of thousands of genes, exquisitely regulated in both spatial and temporal dimensions, to achieve the proper specialization and inter-connectivity of brain regions. Consequently, the dysregulation of complex gene networks in the developing brain is believed to underlie many neurodevelopmental disorders, such as autism spectrum disorders (ASD). Autism has a significant genetic etiology, but there are hundreds of genes implicated, and their functions are heterogeneous and complex. Therefore, an understanding of shared molecular and cellular pathways underlying the development ASD has remained elusive, hampering attempts to develop common diagnostic biomarkers or treatments for this disorder. I hypothesized that analyzing functional genomics relationships among ASD candidate genes during normal human brain development would provide insight into common cellular and molecular pathways that are affected in autistic individuals, and may help elucidate how hundreds of diverse genes can all be linked to a single clinical phenotype. This thesis describes a coordinated set of bioinformatics experiments that first (i) assessed for gene expression and co-expression properties among ASD candidates and other non-coding RNAs during normal human brain development to discover potential shared mechanisms; and then (ii) directly assessed for changes in these pathways in autistic post-mortem brain tissue. The results demonstrated that when examined in the context of normal human brain gene expression during early development, autism candidate genes appear to be strongly related to the neurodevelopmental pathways of synaptogenesis, mitochondrial function, glial cytokine signaling, and transcription/translation regulation. Furthermore, the known sex bias in ASD prevalence appeared to relate to differences in gene expression between the developing brains of males and females. Follow up studies in autistic brain tissue confirmed that changes in mitochondrial gene expression networks, glial pathways, and gene expression regulatory mechanisms are all altered in the brains of autistic individuals. Together, these results show that the heterogeneous set of autism candidate genes are related to each other through shared transcriptional networks that funnel into common molecular mechanisms, and that these mechanisms are aberrant in autistic brains. 4 Acknowledgements This work would not have been possible without the tremendous amount of support I received from many individuals and organizations. First, I would like to thank my graduate program and my two thesis advisors. The National Institutes of Health-University of Cambridge Biomedical Scholars Program has been a tremendous training opportunity for me. I have had great support from both NIH and the University of Cambridge throughout my studies, and I am incredibly grateful to have been given this opportunity. I am especially indebted to Dr. Azim Surani at the University of Cambridge for his guidance and flexibility with me as this work developed away from our original plans, and for his continued mentoring and guidance. To Dr. Owen Rennert at the National Institute of Child Health and Human Development, I will be forever grateful for all that he has done for me. Throughout my training I have been funded by or received awards from a number of organizations that have allowed me to pursue this work. The National Institute of Child Health and Human Development at NIH, the NIH-Oxford/Cambridge Biomedical Scholars Program, the NIH MD/PhD Global Doctoral Partnership program, Baylor College of Medicine Medical Scientist Training Program, the Allen Institute for Brain Science, the American Academy of Neurology, Robinson College, and the University of Cambridge have all provided me with generous support. I would like to thank others with whom I have worked, learned from, and have been given help during my studies, including members of Dr. Surani’s and Dr. Rennert’s laboratories, the administrators at NICHD, the NIH-Oxford/Cambridge Program, Robinson College, and Baylor College of Medicine, and our collaborators at TU Delft. Most importantly, I would like to thank my parents, Nicholas and Lucille Ziats, for their unwavering support, guidance, and inspiration. 5 Table of Contents Publications Resulting From This Work..........................................................................................7 List of Abbreviations .......................................................................................................................8 List of Figures and Tables................................................................................................................9 1 Introduction ...............................................................................................................................12 1.1 Autism Spectrum Disorders .....................................................................................................13 1.2 Functional Genomics of Human Brain Development ..............................................................22 1.3 Previous Functional Genomics Studies of ASD ......................................................................42 1.4 Major Unanswered Questions and Motivations of this Work .................................................46 2 Characterizing ASD Candidate Genes During Normal Human Neurodevelopment .........47 2.1 Expression Profiling of Individual Autism Candidate Genes ..................................................55 2.2 Co-expression Network Analysis of Autism Candidate Genes ...............................................75 2.3 Global Sex Differences in Gene Expression ............................................................................95 2.4 Identification of Differentially Expressed MicroRNAs and their Relationship to ASD .......101 3 Functional Genomics Studies of Autistic Post-mortem Brain Tissue ................................111 3.1 Long Non-coding RNAs are Dysregulated in Autistic Brain ................................................115 3.2 Altered Glial Marker Expression in Autistic Brains ..............................................................126 3.3 Altered Expression of the Mitochondrial Genome in Autism ...............................................136 4 Conclusions 4.1 Summary ................................................................................................................................146 4.2 Future Perspectives ................................................................................................................148 Appendix Theoretical Hypothesis on the Role of the Cerebellum in Autism ..............................................149 Description of Work Performed in Collaboration .......................................................................152 Additional Tables and Figures .....................................................................................................153 References ...................................................................................................................................167 Supplementary Information .....................................................................................................192 6 Publications Resulting From This Work Mahfouz A,* Ziats MN,* Rennert OM, Lelieveldt BP, Reinders MJ. Co-expression Network Analysis of the Developing Human Brain Transcriptome Reveals Shared Pathways among Autism Candidate Genes. Revision Submitted. *equal contribution Edmonson C*, Ziats MN*, Rennert OM. Altered glial marker expression in autistic post- mortem pre-frontal cortex and cerebellum Mol Autism. 2014;5(1):3. *equal contribution Ziats MN, Rennert OM. The cerebellum in autism: pathogenic or an anatomical beacon? Cerebellum. 2013 Oct;12(5):776-7. Ziats MN, Rennert OM. Identification of differentially expressed microRNAs across the developing human brain. Mol Psychiatry. 2013 Aug 6. [Epub ahead of print] Ziats MN, Rennert OM. Sex-biased gene expression in the developing brain: implications for autism spectrum disorders. Mol Autism. 2013 May 7;4(1):10. Ziats MN, Rennert OM. Aberrant expression of long noncoding RNAs in autistic brain. J Mol Neurosci. 2013 Mar;49(3):589-93. Ziats MN, Rennert OM. Expression profiling of autism candidate genes during human brain development implicates central immune signaling pathways. PLoS One. 2011;6(9):e24691. 7 List of Abbreviations Amyg, Amy amygdala ASD autism spectrum disorder ATP adenosine triphosphate BA Broadman’s area CDC Center for Disease Control and Prevention Cere cerebellum CGH comparative
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