A FUNCTIONAL CHARACTERISATION OF THE PCSK6 LOCUS ASSOCIATED WITH HANDEDNESS Robert Shore A Thesis Submitted for the Degree of PhD at the University of St Andrews 2016 Full metadata for this thesis is available in St Andrews Research Repository at: http://research-repository.st-andrews.ac.uk/ Please use this identifier to cite or link to this thesis: http://hdl.handle.net/10023/15719 This item is protected by original copyright This item is licensed under a Creative Commons Licence http://creativecommons.org/licenses/by-nc-nd/4.0/ A functional characterisation of the PCSK6 locus associated with handedness Robert Shore This thesis is submitted in partial fulfilment for the degree of Doctor of Philosophy at the University of St Andrews Date of Submission: December 1 2015 1. Candidate’s declarations: I Robert Shore hereby certify that this thesis, which is approximately 40,000 words in length, has been written by me, and that it is the record of work carried out by me, or principally by myself in collaboration with others as acknowledged, and that it has not been submitted in any previous application for a higher degree. I was admitted as a research student in August 2012 and as a candidate for the degree of PhD in August 2012; the higher study for which this is a record was carried out in the University of St Andrews between 2012 and 2015. 2. Supervisor’s declaration: I hereby certify that the candidate has fulfilled the conditions of the Resolution and Regulations appropriate for the degree of PhD in the University of St Andrews and that the candidate is qualified to submit this thesis in application for that degree. Date …… signature of supervisor ……… 3. 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Where part of a thesis is to be embargoed, please specify the part and the reason. ii Dedicated to the memory of my father, Joseph Shore (1949-2013) Ar dheis Dé go raibh a anam iii Abstract Humans display a 90% population level bias towards right-handedness, implying the vast majority of people have a left-hemisphere dominant for motor control. Although handedness presents a weak, but very consistent heritability across the literature (estimated to be approximately 25%), to date few genetic loci associated with this complex trait have been identified and replicated in subsequent studies. One such gene which has been found to be associated with handedness and subsequently replicated is PCSK6, most recently through a quantitative GWAS (P < 0.5*10-8, Brandler et al. (2013)). Interestingly, PCSK6 is known to activate Nodal, a morphogen involved in a highly conserved bilaterian pathway known to regulate left-right body axis determination. Here I present the first molecular characterisation of a handedness-associated region by conducting a detailed functional analysis of the PCSK6 locus, combining genetic analysis, in silico prediction and molecular assays to investigate how common genetic variants influence handedness-related phenotypes. Specifically, I defined the associated locus to be 12.7 kb in size, spanning a predicted 1.8 kb bidirectional promoter which controls the expression of both an antisense long non-coding RNA (lncRNA), and a novel short PCSK6 isoform. A series of luciferase-expressing constructs were generated to characterise the promoter, identifying a minimal sequence capable of driving transcription in a sense strand direction. I have demonstrated experimentally that one of the top associated markers in previous GWA studies, rs11855145, directly creates/disrupts a suspected transcription factor bind site in the vicinity of this bidirectional promoter. Further functional studies of the genetic variation within PCSK6 may help explain the molecular regulatory mechanisms affecting gene expression. This project provides a model for assays to study other GWAS-nominated candidate genes, and in particular for establishing the role of noncoding variants. The findings from this study support the role of common variants in influencing complex phenotypes, such as handedness. Thesis word count: 40,000 words iv Acknowledgements I would like to thank my supervisor Dr Silvia Paracchini for giving me not only the opportunity to come to St Andrews and complete a PhD in a topic I found enjoyable and intriguing in equal measure, but also for the advice and support you provided and the independence you allowed when required. For that I will always be grateful. I am indebted to my thesis committee; Professor Lindsey Kent and Professor Terry Smith who helped with their encouragement, ideas and patience; I am in no doubt how compromised my endeavours would have been without your help. A sincere appreciation also goes to Dr Catherine Botting for her mass spectrometry services, Dr Kerry Pettigrew for her toils in helping define the clinical cohorts and my internal and external examiners Dr Matt Holden and Dr Nick Bray for giving up so much of their time to improve this thesis. Finally, I would like to thank my parents, my family and my friends Liam Scally and Aleksandra Sobota for their infuriatingly persistent refusal to ever let me give up. v Table of Contents Abstract iv Acknowledgements v Table of Contents vi List of Figures xi List of Tables xiv Abbreviations xvi 1 Introduction 1 1.1 Handedness .............................................................................................................. 1 1.1.1 What is handedness? ...................................................................................... 1 1.1.2 Limb preference in the Animal Kingdom ....................................................... 2 1.1.3 Handedness in non-human primates ............................................................... 5 1.1.4 Right-hand dominance in the hominin lineage ............................................... 6 1.1.5 How did handedness evolve? ......................................................................... 7 1.1.6 Handedness and Language ............................................................................. 8 1.1.7 Why study handedness? ............................................................................... 10 1.1.8 The development of handedness .................................................................. 12 1.1.9 Genetic models of handedness ..................................................................... 13 1.1.10 Previous studies and candidate genes ........................................................... 14 1.2 PCSK6 ................................................................................................................... 22 1.2.1 The PCSK6 gene ......................................................................................... 22 1.2.2 PCSK6 role in complex traits and disease .................................................... 23 1.2.3 Nodal signalling pathway and cilia .............................................................. 24 1.2.4 RD candidate genes and cilia ....................................................................... 27 1.2.5 Role of regulatory elements during neuronal development ........................... 27 1.2.5.1 Non-coding RNA 28 vi 1.2.5.2 VNTRs 29 1.2.5.3 Bidirectional promoters 31 1.3 Conclusion ............................................................................................................. 32 1.3.1 Overall aim of this thesis ............................................................................. 32 1.3.2 Overview of the study methodology ............................................................ 33 2 ALSPAC Cohort Association Analysis 35 2.1 Abstract ................................................................................................................