UNIVERSITY OF CATANIA INTERNATIONAL PhD PROGRAM IN NEUROSCIENCE XXIX Cycle VALENTINA LA COGNATA READING THROUGH THE BUILDING BLOCKS OF THE GENOME: EXONIC VARIATION IN PARKINSON'S DISEASE PhD Thesis Coordinator: Supervisors: Prof. Salvatore Salomone Prof. Velia D’Agata Dr. Sebastiano Cavallaro January 2017 Copyright © V. La Cognata, 2017 All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means, without prior permission of the author. The copyright of the published papers remains with the publishers. SUMMARY Abstract ...................................................................................................... 5 Chapter 1 .................................................................................................... 7 PARKINSON’S DISEASE ....................................................................................................... 9 HAS EXONIC VARIATION A ROLE IN PD? ............................................................................12 AIMS OF THE PhD WORK ...................................................................................................13 Chapter 2 .................................................................................................. 15 ABSTRACT ........................................................................................................................17 INTRODUCTION ................................................................................................................18 CNVs: A PREVALENT SOURCE OF GENOMIC VARIATIONS ...................................................20 COPY NUMBER VARIATIONS IN FAMILIAR PD GENES .........................................................24 SNCA .................................................................................................................................................. 24 PARK2................................................................................................................................................ 29 PINK1 ................................................................................................................................................ 31 PARK7................................................................................................................................................ 32 ATP13A2 ........................................................................................................................................... 33 THE 22q11.2 DELETION ....................................................................................................33 HIGH-THROUGHPUT WHOLE-GENOME STUDIES TO MAP CNVs IN PD ................................35 A SYSTEMS BIOLOGY APPROACH FOR RARE AND SINGLETON CNVs ...................................37 CONCLUSIONS ...................................................................................................................45 Chapter 3 .................................................................................................. 59 ABSTRACT ........................................................................................................................61 INTRODUCTION ................................................................................................................62 MATERIALS AND METHODS ..............................................................................................64 GENE SELECTION AND aCGH DESIGN STRATEGY ........................................................................ 64 CLINICAL SAMPLES SELECTION ..................................................................................................... 65 MICROARRAY EXPERIMENT AND DATA ANALYSIS ..................................................................... 65 VALIDATION ..................................................................................................................................... 66 RESULTS ...........................................................................................................................67 aCGH DESIGN ON A TARGETED PD GENE PANEL ......................................................................... 67 CNVs OF PD-RELATED GENES DETECTED THROUGH THE NEUROARRAY PLATFORM ........... 70 DISCUSSION ......................................................................................................................75 CONCLUSIONS ...................................................................................................................77 Chapter 4 .................................................................................................. 85 1 ABSTRACT ........................................................................................................................87 INTRODUCTION ................................................................................................................88 GENETICS OF PARKINSON’S DISEASE ................................................................................90 AUTOSOMAL DOMINANT PD GENES ..................................................................................91 SNCA .................................................................................................................................................. 91 LRRK2 ................................................................................................................................................ 94 VPS35 ................................................................................................................................................ 95 AUTOSOMAL RECESSIVE PD GENES ...................................................................................97 EARLY- ONSET TYPICAL PD GENES ............................................................................................. 97 JUVENILE ATYPICAL PD GENES .................................................................................................. 101 X-LINKED PARKINSONISM ............................................................................................... 104 OTHER PD-RELATED GENES ............................................................................................ 106 SNCAIP ............................................................................................................................................. 106 MAPT ............................................................................................................................................... 107 GBA .................................................................................................................................................. 108 GENOME-WIDE RNA EXPRESSION ANALYSIS REVEALS GLOBAL ALTERNATIVE SPLICING CHANGES IN PD ............................................................................................................... 111 THE ROLE of miRNA AND lncRNA in PD ALTERNATIVE SPLICING MODULATION .............. 112 CONCLUSIONS ................................................................................................................. 113 Chapter 5 ................................................................................................ 125 ABSTRACT ...................................................................................................................... 127 INTRODUCTION .............................................................................................................. 128 ALTERNATIVE SPLICING, BASIC CONCEPTS ..................................................................... 129 ALTERNATIVE SPLICING OF PARK2 ................................................................................. 131 HUMAN PARK2 ALTERNATIVE SPLICE VARIANTS ..................................................................... 131 RAT Park2 ALTERNATIVE SPLICE VARIANTS ............................................................................ 135 MOUSE Park2 ALTERNATIVE SPLICE VARIANTS ....................................................................... 137 SPECIES-SPECIFIC ALTERNATIVE SPLICING OF PARK2 IN HUMAN, RAT AND MOUSE ........ 139 ALTERNATIVE SPLICING OF PARK2 PRODUCES DIVERSITY .............................................. 140 ALTERNATIVE SPLICING OF PARK2 IS TISSUE AND CELL SPECIFIC ................................... 142 CONCLUSIONS ................................................................................................................. 147 Chapter 6 ................................................................................................ 153 ABSTRACT ...................................................................................................................... 155 INTRODUCTION .............................................................................................................. 156 PARK2 ALTERNATIVE SPLICE TRANSCRIPTS PRODUCE ISOFORMS WITH DIFFERENT STRUCTURES AND FUNCTIONS ........................................................................................ 162 2 Reading through the building blocks of the genome: exonic variation in PD EVIDENCES OF MULTIPLE PARKIN ISOFORMS IN BRAIN .................................................. 163 THE DIVERSIFIED PANEL OF ANTIBODIES COMMERCIALLY AVAILABLE AGAINST PARK2 . 167 CONCLUSION .................................................................................................................. 177 Chapter 7 ................................................................................................ 183 GENERAL DISCUSSION ...................................................................................................