SEGMENTAL DUPLICATIONS PROMOTE GENOMIC INSTABILITY IN HUMAN CHROMOSOME 15q11-q13 by DEVIN PAUL LOCKE Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Dr. Evan E. Eichler Department of Genetics CASE WESTERN RESERVE UNIVERSITY August, 2004 Copyright © 2004 by Devin Paul Locke All rights reserved CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. To Patricia Anne Clark and Paul Arthur Locke, otherwise known as Mom and Dad, for their unwavering support. TABLE OF CONTENTS List of Figures................................................................................................................... ix List of Tables .................................................................................................................... xi Abstract............................................................................................................................ xii Acknowledgements ........................................................................................................ xiv Chapter 1: Introduction and Objectives......................................................................... 1 The Landscape of the Human Genome........................................................................... 2 Introduction................................................................................................................. 2 Repetitive Sequences of the Mammalian Genome ......................................................... 3 Structural DNA ........................................................................................................... 3 Functionless Junk: LINEs and SINEs and HERVs..................................................... 4 Segmental Duplications .................................................................................................. 5 Defining Segmental Duplication................................................................................. 5 The Initial Description of a Segmental Duplication ................................................... 8 Identifying Segmental Duplications ........................................................................... 9 How Segmental Duplications Arise.......................................................................... 12 Pericentromeric Regions............................................................................................... 12 Structural Variation Among Pericentromeric Regions ............................................. 12 The Two-Step Model of Pericentromeric Duplication ............................................. 15 Paralogous Sequence Variants.................................................................................. 17 Subtelomeric Regions ................................................................................................... 20 Evolution by Duplication.............................................................................................. 22 Local Gene Duplication ............................................................................................ 22 Whole-Genome Duplication ..................................................................................... 23 Tandem Duplications................................................................................................ 25 Chromosome Evolution ............................................................................................ 26 Primate Genome Variation ........................................................................................... 28 Karyotype Variation.................................................................................................. 28 Nucleotide Variation................................................................................................. 29 Array Comparative Genomic Hybridization................................................................. 31 Chromosome 15q11-q13 Exemplifies Genomic Plasticity........................................... 37 Genomic Disorders Within 15q11-q13: Prader-Willi and Angelman Syndromes ... 38 Imprinting Implicated in PWS/AS............................................................................ 39 Duplications and 15q11-q13 Genomic Rearrangements .......................................... 40 v Timing of 15q11-q13 Structural Evolution............................................................... 42 The Diversity of Additional 15q11-q13 Rearrangements......................................... 43 Phenotypically Silent 15q11 Variation ..................................................................... 44 Research Objectives...................................................................................................... 45 Chapter 2: Large-scale Variation Among Human and Great Ape Genomes Determined by Array Comparative Genomic Hybridization .................................... 47 Abstract......................................................................................................................... 48 Introduction................................................................................................................... 49 Results........................................................................................................................... 51 Discussion..................................................................................................................... 65 Materials and Methods.................................................................................................. 68 Human BAC Arrays.................................................................................................. 68 Primate DNA Samples.............................................................................................. 69 Array Comparative Genomic Hybridization............................................................. 70 Fluorescence In Situ Hybridization........................................................................... 71 BAC Analysis ........................................................................................................... 72 Acknowledgements....................................................................................................... 73 Chapter 3: Refinement of a Chimpanzee Pericentric Inversion Breakpoint to a Segmental Duplication Cluster ...................................................................................... 74 Abstract......................................................................................................................... 75 Introduction................................................................................................................... 76 Results........................................................................................................................... 77 Discussion..................................................................................................................... 87 Materials and Methods.................................................................................................. 93 FISH Probe Selection................................................................................................ 93 Comparative FISH .................................................................................................... 94 Duplication Analysis................................................................................................. 94 Southern Analysis ..................................................................................................... 95 Acknowledgements....................................................................................................... 96 Chapter 4: Molecular Evolution of the Human Chromosome 15 Pericentromeric Region............................................................................................................................... 98 Abstract......................................................................................................................... 99 Introduction................................................................................................................. 100 Results and Discussion ............................................................................................... 102 In Silico Analysis of the 15q11 Pericentromeric Region........................................ 102 vi FISH Analysis of Human 15q11............................................................................. 109 Comparative FISH Analysis of Non-human Primate Chromosomes ..................... 112 Phylogenetic Analysis............................................................................................. 115 Validation of the 15q11 Assembly ......................................................................... 124 STS Analysis........................................................................................................... 125 BAC End Sequence Analysis.................................................................................