Investigation of Gene Splicing Variants Contributing Risk to Psychiatric Disorders by Emma Reble A thesis submitted in conformity with the requirements for the degree of Master of Science Institute of Medical Science University of Toronto © Copyright by Emma Reble 2017 Investigation of Gene Splicing Variants Contributing Risk to Psychiatric Disorders Emma Reble Master of Science Institute of Medical Science University of Toronto 2017 Abstract The majority of the variants found through genome-wide association studies (GWAS) to be associated with psychiatric disorders are not found to cause a protein coding change, therefore it is predicted that these variants are causing gene expression changes, including changes to alternative splicing. Datasets of variants associated with alternative splicing changes in the general population have also been curated with the aim of aiding the search for functional GWAS variants. This study uses these datasets to identify variants associated with alternatively spliced isoforms in schizophrenia and bipolar disorder. Alternatively spliced isoforms in four genes (APOPT1, AS3MT, NEK4 and RPGRIP1L) were identified that converge on the mitochondria and cilia, implicating these cellular structures in psychiatric disease and providing avenues of genes and pathways for further study. A method to study alternative splicing changes in psychiatric disorders is presented that would also be a useful tool for studying other complex genetic disorders. ii Table of Contents Table of Contents ............................................................................................................. iii Acknowledgments ............................................................................................................ vi Statement of Contributions ............................................................................................ vii List of Abbreviations ..................................................................................................... viii List of Tables ..................................................................................................................... x List of Figures ................................................................................................................... xi Chapter 1: Introduction and Background ...................................................................... 1 1.1 Psychiatric Disorders and Their Genetic Contributions ...................................... 1 1.1.1 Schizophrenia ................................................................................................................. 1 1.1.2 Bipolar Disorder ............................................................................................................. 3 1.1.3 Elucidating Genetic Risk for Psychiatric Disorders ................................................... 4 1.2 Gene Splicing ............................................................................................................ 5 1.2.1 Splicing Mechanisms ..................................................................................................... 5 1.2.2 Types of Alternative Splicing ........................................................................................ 7 1.2.3 Alternative Splicing in Mendelian Disease .................................................................. 9 1.2.4 Alternative Splicing in the Brain ................................................................................ 10 1.2.5 Alternative Splicing in Psychiatric Disorders ........................................................... 11 1.2.5.1 Alternative Splicing in Autism Spectrum Disorder (ASD) .................................... 12 1.2.5.2 Alternative Splicing in Bipolar Disorder ................................................................ 14 1.2.5.3 Alternative Splicing in Schizophrenia .................................................................... 15 1.3 Splicing Predictions and Datasets ........................................................................ 17 1.3.1 SPIDEX ......................................................................................................................... 18 1.3.2 Altrans ........................................................................................................................... 19 1.3.3 sQTLSeekeR ................................................................................................................. 19 1.3.4 Protein Truncating Variants (PTV) ........................................................................... 20 1.3.5 DLPFC sQTLs .............................................................................................................. 21 1.4 Protein Structure and Function Prediction ......................................................... 21 1.5 Hypothesis, Aims and Overview of Thesis ........................................................... 23 Chapter 2: Materials and Methods ............................................................................... 25 2.1 Identification of Schizophrenia and Bipolar Disorder Candidate SNVs .......... 25 2.2 Brain tissue samples ............................................................................................... 25 2.3 SNV Genotyping .................................................................................................... 27 2.4 Expression of Alternative Splicing Isoforms ....................................................... 28 2.4.1 Identification of Splicing Variants ............................................................................. 28 2.4.2 Quantification of Identified Splicing Variants Using ddPCR .................................. 31 2.5 Predicting the Mechanism of the Splice Variants ............................................... 33 iii 2.6 Predicting the Effect on Protein Function ........................................................... 34 Chapter 3: Results ........................................................................................................... 35 3.1 Splicing SNVs associated with Schizophrenia and Bipolar Disorder ............... 35 3.2 Genes studied for alternative splicing .................................................................. 42 3.2.1 APOPT1 ......................................................................................................................... 42 3.2.1.1 APOPT1 SNVs Associated with Alternative Splicing ........................................... 43 3.2.1.2 Identification of Two APOPT1 Transcript Isoforms .............................................. 45 3.2.1.3 Quantification of APOPT1 Isoforms and Association to rs4906337, rs10431750 and rs7140568 ........................................................................................................................ 46 3.2.1.4 Protein Function Prediction of APOPT1 Isoforms ................................................. 48 3.2.2 AS3MT ........................................................................................................................... 50 3.2.2.1 AS3MT SNV Predicted to Alter Splicing ............................................................... 51 3.2.2.2 Identification of Two AS3MT Transcript Isoforms ................................................ 53 3.2.2.3 Quantification of AS3MT Isoforms and Association to rs3740400 ........................ 54 3.2.2.4 Protein Function Prediction of AS3MT Isoforms ................................................... 56 3.2.2.5 Hypothesis of inefficient arsenic metabolism contributing to risk of schizophrenia 57 3.2.3 BNIP3L .......................................................................................................................... 59 3.2.3.1 SNVs Associated with Alternative BNIP3L Transcripts ........................................ 59 3.2.3.2 Identification of Two BNIP3L Transcript Isoforms ............................................... 60 3.2.3.3 Quantification of BNIP3L Isoforms and Association to rs11779570, rs11786753, rs60377146 and rs11135938 .................................................................................................. 61 3.2.3.4 Protein Function Prediction of BNIP3L Isoforms .................................................. 63 3.2.4 CACNA1D ..................................................................................................................... 65 3.2.5 KIF21B .......................................................................................................................... 66 3.2.6 LRRC48 ......................................................................................................................... 68 3.2.6.1 LRRC48 SNVs Predicted to Alter Exon 7 and 8 Splicing ...................................... 69 3.2.6.2 LRRC48 SNV Predicted to Alter Exon 2 Splicing ................................................. 71 3.2.6.3 Identification of Three LRRC48 Transcript Isoforms ............................................. 72 3.2.6.4 Quantification of LRRC48 Isoforms and Association to rs11870660 .................... 73 3.2.6.5 Protein Function Prediction of LRRC48 Isoforms .................................................. 76 3.2.7 NEK4 ............................................................................................................................. 76 3.2.7.1 NEK4 SNVs Predicted to Alter Exon 5 Splicing .................................................... 77 3.2.7.2 NEK4 SNVs Predicted to Alter Exon 4 Splicing .................................................... 78 3.2.7.3