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Download The EXPLORING MITOCHONDRIAL DNA ABNORMALITIES IN HIV-EXPOSED UNINFECTED CHILDREN DIAGNOSED WITH AUTISM SPECTRUM DISORDER: A CASE CONTROL STUDY by Matthew Budd B.A., Mount Royal University, 2012 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pathology and Laboratory Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2016 © Matthew Budd, 2016 Abstract Background: Antiretroviral therapy has reduced mother-to-child HIV transmission from 25- 40% to less than 2%. Thus, increasing numbers of HIV-exposed uninfected (HEU) children are being born with perinatal exposure to antiretrovirals. Recently, a Canadian HIV clinic noticed a high prevalence of autism spectrum disorder (ASD) in HEU children. This prompted our analysis of HEU children enrolled in the pan-Canadian Children & Women AntiRetrovirals & Markers of Aging (CARMA) cohort study. Significant differences in mitochondrial DNA to nuclear DNA ratio (mtDNA content) have been observed in ASD children and HEUs as a potential marker for mitochondrial dysfunction, which has been theorized as a possible mechanism underlying abnormal neurodevelopment. We hypothesized that HEU children with ASD would have significantly different leukocyte mtDNA content than HEU children without ASD and/or HUU children with and without ASD. Methods: CARMA HEU children with ASD (n=14) were matched 1:3 on age, sex, and ethnicity to HEU children without ASD (n=42), HUU anonymous controls (n=42), and HUU children with ASD in the BC Autism Spectrum Interdisciplinary Research (ASPIRE) program (n=42). Non-ASD HUU siblings of ASD children (n=9) were also studied and grouped with the HUU controls for the purposes of analyses (n=51 total). MtDNA content was assessed using qPCR. Results: Among 299 HEU children in CARMA, 14 (4.7%) were diagnosed with ASD, substantially (>3-fold) above North American prevalence estimates (1.5%). ii HEU children with ASD had higher mtDNA content (median[interquartile range]: 163[150– 179]) than non-ASD HEUs (115[91–153], p=0.02), HUUs with ASD (110[99–132], p=0.0001), and HUU controls (100[73–121], p<0.0001). Non-autistic HEU children and ASD children with no HIV/cART exposure had higher mtDNA content than controls (p=0.004 and p=0.03, respectively), but did not significantly differ from each other (p=0.2). Conclusions: Our results suggest a possible cumulative association between elevated leukocyte mtDNA content and both HEU and ASD status. This may implicate mitochondrial dysfunction as a contributor to the high ASD prevalence in our cohort. It is unclear if this effect is modulated by exposure to antiretrovirals or maternal HIV but it is consistent with studies suggesting increased mtDNA content as an adaptive mechanism to mitochondrial dysfunction. iii Preface The contents of this thesis are my own original work. The experiments were designed by me in conjunction with my supervisor, Dr. Hélène Côté, and Dr. Jason Brophy of the Children’s Hospital of Eastern Ontario. All conducted research was previously approved by the University of British Columbia Research Ethics Board (H08-02018). The collaborative use of clinical samples from the BC Autism Spectrum Interdisciplinary Program was approved August 5, 2015 by the University of British Columbia Research Ethics Board as an amendment to the original protocol (H08-02018-A024). Written parental consent was received for all enrolled participants, and informed assent was sought from those participants with the capacity to provide it. A copy of the consent form is attached in the appendix. A version of this study is currently being prepared in manuscript format to be published before the completion of my graduate program. A manuscript describing the monochrome, multiplex method of mtDNA quantitation is currently in preparation. I conducted most of the measurements described in Chapters 3 and 4, organized the data and matching, and performed all statistical analyses. iv Table of Contents Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iv Table of Contents ...........................................................................................................................v List of Tables ..................................................................................................................................x List of Figures .............................................................................................................................. xii List of Abbreviations ................................................................................................................. xiv Acknowledgements .................................................................................................................. xviii Dedication .....................................................................................................................................xx Chapter 1: Introduction ................................................................................................................1 1.1 Introduction to thesis....................................................................................................... 1 1.2 Human immunodeficiency virus (HIV) .......................................................................... 3 1.2.1 Origin and transmission .............................................................................................. 3 1.2.2 Epidemiology .............................................................................................................. 3 1.2.3 Morphology and genome structure ............................................................................. 4 1.2.4 Replication cycle ......................................................................................................... 7 1.2.5 Immunopathogenesis and stages of infection ............................................................. 9 1.2.6 Combination antiretroviral therapy (cART) ............................................................. 11 1.2.6.1 Classes of cART ............................................................................................... 12 1.2.6.1.1 Nucleoside reverse transcriptase inhibitors ................................................. 12 1.2.6.1.2 Non-nucleoside reverse transcriptase inhibitors ......................................... 15 1.2.6.1.3 Protease inhibitors ....................................................................................... 15 v 1.2.6.1.4 Integrase inhibitors ...................................................................................... 16 1.2.6.1.5 Fusion and entry inhibitors .......................................................................... 17 1.2.7 Guidelines for HIV treatment and prevention .......................................................... 18 1.2.7.1 In Canada .......................................................................................................... 18 1.2.7.2 In the developing world .................................................................................... 19 1.2.7.3 HIV and cART in pregnancy ............................................................................ 21 1.3 Mitochondria ................................................................................................................. 24 1.3.1 Structure and function ............................................................................................... 24 1.3.1.1 The electron transport chain and oxidative phosphorylation ............................ 25 1.3.2 Mitochondrial DNA (mtDNA) ................................................................................. 27 1.3.2.1 Replication ........................................................................................................ 28 1.3.2.2 ROS-induced oxidative damage ....................................................................... 29 1.3.2.3 Maintenance ...................................................................................................... 30 1.4 Autism spectrum disorder (ASD) ................................................................................. 30 1.4.1 Diagnostic criteria and instrumentation .................................................................... 31 1.4.1.1 DSM-V .............................................................................................................. 31 1.4.1.2 Screening and assessment ................................................................................. 33 1.4.1.3 ADOS-G and ADI-R......................................................................................... 34 1.4.2 Demographics ........................................................................................................... 35 1.4.3 Other factors associated with ASD ........................................................................... 35 1.5 ASD, HIV, cART, and mitochondrial dysfunction ....................................................... 37 Chapter 2: Study design and participant demographics .........................................................39 2.1 Recruitment ................................................................................................................... 39 vi 2.2 Eligibility and selection criteria
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