Identification and characterisation of rare CACNG5 genetic variants in bipolar disorder and schizophrenia Thesis submitted for the degree of Doctor of Philosophy UCL Yi-Chun Lin Molecular Psychiatry Laboratory University College London 2010 – 2015 1 I, Yi-Chun Lin, confirm that all the work presented in this thesis is my own. I confirm that the information has been derived from other sources; it has been indicated in the thesis. 2 Acknowledgements This thesis would not have been completed without the help and support of colleagues and friends to whom I would now like to express my sincere thanks First and foremost of my thanks goes to my supervisor Dr Andrew McQuillin, who gave me this wonderful and exciting opportunity to carry out this PhD. His encouragement has provided tremendous support throughout my PhD. I am extremely grateful for his insightful advice, patience and kindness. I would also like to express my appreciation and thanks to Professor Hugh Gurling, my second supervisor, who provided further guidelines and support to enable me to complete this PhD research. Very special thanks to Dr. Radhika Kandaswamy and Dr. Sally Sharp, who have been guiding me with infinite patience in my experimental studies. I am indebted to them for their enthusiasm and advice, their friendship and support leading to the completion of research experiments. Also I would like to thank all my colleagues from my lab Michael, John, Alessia for their support. Finally, I would also like to thank my family, especially my parents for their financial support and my friends for their encouragement and support. 3 Abstract Schizophrenia (SCZ) and bipolar disorder (BPD) are common, highly heritability psychiatric disorders. Genome-wide association studies have found evidence of shared genetic susceptibility to both diseases. The most notable example is CACNA1C which encodes the 1 subunit of L-type calcium channels. Several other calcium channel genes have also been implicated in BPD and/or SCZ and together there is support for a role for these genes in both diseases. The primary function of several subunit calcium channel genes appears to be the regulation of AMPA receptor localisation and function. Collectively these are known as Transmembrane AMPA receptor Regulatory Proteins (TARPs). This thesis aimed to identify disease relevant genetic variation in one such TARP, CACNG5, and to study the effect of these variants. CACNG5 variants in the exons and promoter region were identified in 1098 BPD, 618 SCZ, and 1087 control individuals. Four novel non-synonymous SNPs (nsSNPs) and four nsSNPs were identified. Burden analysis of nsSNPs in BPD and SCZ found evidence for association (p=0.0022). This association was strengthened by inclusion of data from European samples in the 1000 Genomes project (p=0.00057). However, combined data with the UK10K and Swedish exome sequence studies founds a weakened association signal (p=0.0082). Functional analyses using co-expression of AMPAR2 and CACNG5 constructs containing the eight nsSNPs were used to analyse changes in the expression and/or trafficking of 5 and AMPA receptors. Four of the variants were associated with decreased AMPAR2 expression as a consequence of altered trafficking to the cell surface. V146M (identified in 2 SCZ patients) overexpression increased AMPAR2 trafficking to the cell surface (p<0.005); conversely, T164L (identified in one SCZ patient) overexpression decreased the expression 4 of AMPAR2 and its cell surface trafficking (p<0.05). Our results suggest a role for CACNG5 variants in SCZ and/or BPD and that this may be mediated via dysregulation of AMPARs. 5 Table of Contents Acknowledgements ................................................................................................................... 3 Abstract ..................................................................................................................................... 4 Table of Contents ...................................................................................................................... 6 Table of Tables ....................................................................................................................... 11 Table of Figures ...................................................................................................................... 14 1 Introduction ..................................................................................................................... 28 1.1 Psychiatric disorders ................................................................................................ 28 1.1.1 Overview ........................................................................................................... 28 1.1.2 History of psychiatric disorders ........................................................................ 29 1.1.3 Diagnosis of psychiatric disorders .................................................................... 31 1.1.3.1 Diagnosis of bipolar disorder ............................................................................ 33 1.1.4 Epidemiology similarities in BPD and SCZ ..................................................... 37 1.1.5 Treatment .......................................................................................................... 40 1.1.6 Pathophysiology of BPD and SCZ ................................................................... 44 1.1.7 Heritability of BPD and SCZ ............................................................................ 46 1.2 Molecular Genetics Studies ...................................................................................... 49 1.2.1 Factors affecting genetic studies ....................................................................... 51 1.2.2 Genetic Markers ................................................................................................ 53 1.2.3 Linkage analysis................................................................................................ 54 1.2.4 Association studies............................................................................................ 58 1.2.5 Summary ........................................................................................................... 62 1.2.6 Genome-wide association studies ..................................................................... 63 1.2.7 Chromosomal Rearrangements and CNV ......................................................... 74 1.3 Voltage-gated calcium channel (VGCC) ................................................................. 78 1.3.1 Ca2+ channel γ subunits ..................................................................................... 80 1.4 Glutamate receptors.................................................................................................. 85 6 1.4.1 AMPA receptors ............................................................................................... 87 1.4.2 Trafficking of AMPARs at synapses ................................................................ 95 1.4.3 TARPs as auxiliary protein of AMPARs .......................................................... 96 1.4.4 Trafficking of AMPARs associated with neuro-disorders .............................. 106 2 Aims of the thesis.......................................................................................................... 107 3 MATERIALS AND METHODS .................................................................................. 108 3.1 General Materials and Methods ............................................................................. 108 3.1.1 Research participants – UCL Research sample .............................................. 108 3.1.2 DNA Quantification ........................................................................................ 109 3.2 Variant detection and genotyping .......................................................................... 110 3.2.1 High Resolution Melting Curve Genotyping Assay ....................................... 111 3.2.2 Polymerase Chain Reaction (PCR) ................................................................. 119 3.2.3 Agarose gel electrophoresis ............................................................................ 124 3.2.4 PCR purification ............................................................................................. 125 3.2.5 DNA sequencing ............................................................................................. 127 3.2.6 KASPar Genotyping Assay............................................................................. 131 3.2.7 Association study – burden analysis ............................................................... 142 3.2.8 Bioinformatical analysis ................................................................................. 143 3.3 Preparation of CACNG5 and AMPAR2 Constructs ............................................... 144 3.3.1 Plasmid vectors ............................................................................................... 146 3.3.2 Sub-cloning of CACNG5 inserts into pCMV-Tag4 vectors via traditional restriction sites .............................................................................................................. 154 3.3.3 Sub-cloning of CACNG5 inserts into pTagRFP-C vector via PCR amplification ……………………………………………………………………………….158 3.3.4 Mutating the CACNG5 gene with each variant individually .......................... 161 3.3.5 Large-scale preparation of plasmid DNA ......................................................