Molecular Genetics of Bipolar Disorder Thesis submitted for the degree of Doctor of Philosophy UCL Radhika Kandaswamy Molecular Psychiatry Laboratory University College London 2007 – 2012 1 I, Radhika Kandaswamy, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract Bipolar affective disorder has a strong genetic heritability. In the UCL laboratory, a locus on chromosome 1p36 was found to be linked to bipolar and related unipolar affective disorders with a log of the odds score above 3.00. This region was subjected to fine mapping using tests of allelic association in a case-control sample as part of this thesis in order to identify the genes involved in bipolar disorder. In addition, a GWAS was also employed to fine map other bipolar affective disorder susceptibility genes. The tests of allelic association found evidence for the involvement of the PRKCZ gene. Markers D1S243 and rs3128396 at the PRKCZ gene were significantly associated with bipolar disorder with empirical P = 0.037 and P = 0.040, respectively. Other loci encoding brain expressed proteins found to be associated in the UCL GWAS sample were the genes - GRM3 and GRM7. Therefore, these genes were sequenced using PCR-based genomic sequencing. A 3'-UTR base pair change (rs56173829) in the GRM7 gene was found to be significantly associated with the disorder, although the minor allele was more frequent in controls. A base pair mutation (rs148754219) was found in the GRM3 exon 1 two bases before the translation start codon (forming part of Kozak sequence) of a GRM3 receptor isoform. The mutation was associated with bipolar disorder (P = 0.0046, odds ratio 4.2 (95% CI 1.42-12.37). Transcription factor binding assays and cloning experiments comparing the gene expression activity of wild-type and mutant alleles showed that the mutant allele strongly affected the reporter gene activity in SH- SY5Y and HEK293 cells. If the GRM3 Kozak sequence mutation is confirmed as an 3 important mutation in the aetiology of bipolar disorder, then it could be used as a marker for personalised treatment for a genetic subtype of affective disorders. 4 Table of Contents ABSTRACT ................................................................................................................. 3 TABLE OF CONTENTS ................................................................................................ 5 ACKNOWLEDGEMENTS ............................................................................................ 11 LIST OF FIGURES ..................................................................................................... 13 LIST OF TABLES ...................................................................................................... 16 PUBLICATIONS RELATED TO THIS THESIS ................................................................. 18 ABBREVIATIONS ...................................................................................................... 19 CHAPTER 1 .......................................................................................... 26 GENERAL INTRODUCTION ....................................................................................... 26 1.1. Bipolar disorder.................................................................................................... 27 1.1.1. Overview .............................................................................................. 27 1.1.2. History of Bipolar disorder .................................................................. 28 1.1.3. Epidemiology of Bipolar disorder ........................................................ 30 1.1.4. Diagnosis of Bipolar disorder .............................................................. 34 1.1.5. Treatment of Bipolar disorder .............................................................. 36 1.1.6. Pathophysiology of Bipolar disorder ................................................... 38 1.1.7. Heritability of Bipolar disorder ............................................................ 41 1.1.7.1. Family studies ............................................................................... 41 1.1.7.2. Twin and adoption studies ............................................................ 43 1.2. Molecular genetic studies of Bipolar disorder ................................................... 44 1.2.1 Factors affecting genetic studies ........................................................... 45 1.2.2. Genetic markers ................................................................................... 48 1.2.3. Methods of linkage analysis ................................................................. 48 1.2.4. Linkage findings in Bipolar disorder ................................................... 52 1.2.4.1. Summary of linkage studies at 1p locus ........................................ 58 1.2.5. Candidate gene association studies of Bipolar disorder ....................... 61 1.2.6. Genome-wide association studies in Bipolar disorder ......................... 65 1.2.6.1. Findings of GWAS ........................................................................ 67 1.2.7. CNVs in Bipolar disorder .................................................................... 75 1.3. Glutamate receptors ............................................................................................. 80 5 1.3.1. Ionotropic glutamate receptors ............................................................. 81 1.3.2. Metabotropic glutamate receptors ........................................................ 82 1.3.2.1. Group I mGluRs ............................................................................ 86 1.3.2.2. Group II mGluRs .......................................................................... 88 1.3.2.3. Group III mGluRs ......................................................................... 91 CHAPTER 2 .......................................................................................... 97 AIMS OF THE THESIS ............................................................................................... 97 CHAPTER 3 ........................................................................................ 100 MATERIALS AND METHODS .................................................................................. 100 3.1. General methods ................................................................................................. 101 3.1.1. Research subjects ............................................................................... 101 3.1.1.1. UCL bipolar research sample ...................................................... 101 3.1.1.2. UCL schizophrenia research sample ........................................... 102 3.1.1.3. UCL alcohol dependence research sample ................................. 103 3.1.2. DNA extraction .................................................................................. 104 3.1.2.1. DNA extraction from blood samples .......................................... 104 3.1.2.2. DNA extraction from saliva samples .......................................... 106 3.1.3. DNA quantification ............................................................................ 107 3.1.4. Polymerase chain reaction.................................................................. 108 3.1.4.1. Primer design guidelines ............................................................. 108 3.1.4.2. General PCR principle ................................................................ 109 3.1.4.3. General PCR optimisation........................................................... 110 3.1.4.4. Detection of PCR products using agarose gel electrophoresis.... 113 3.1.4.5. Purification of general PCR procucts .......................................... 114 3.1.5. DNA Sequencing ............................................................................... 115 3.1.5.1. Big Dye sequencing reaction ...................................................... 115 3.1.5.2. Purification of extension products from sequencing PCR .......... 117 3.1.5.3. Analysis of sequencing data ........................................................ 118 3.1.6 Genotyping of genetic markers ........................................................... 119 3.1.6.1. Genotyping of microsatellite markers ......................................... 119 3.1.6.2. SNP genotyping .......................................................................... 123 3.1.6.2.1. KASPar genotyping assay .......................................................................... 123 6 3.1.6.2.2. HRM genotyping assay .............................................................................. 128 3.1.6.2.3. Bioinformatic analysis of novel SNPs ........................................................ 132 3.1.7. GWAS data ........................................................................................ 132 3.1.8. Imputation using 1000 genomes data ................................................. 132 3.1.9. Association and haplotypic analysis programs .................................. 134 3.1.9.1. Haplotypic and statistical analysis for microsatellite markers .... 134 3.1.9.2. Haplotypic and statistical analysis for SNP markers .................. 135 3.1.9.3. Genetic power calculation for replication analysis ..................... 136 3.2. Methods for Chapter 4. Fine mapping of a new bipolar affective disorder locus on chromosome 1p36 ......................................................................................