Contribution of Copy Number Variants to the Risk of Sporadic Congenital Heart Disease A thesis presented by Rachel Soemedi to the Faculty of Medical Sciences in partial fulfilment of the requirements for the degree of Doctor of Philosophy Newcastle University Institute of Genetic Medicine Newcastle upon Tyne, United Kingdom April 2012 Abstract Abstract Congenital heart disease (CHD) is the most common congenital malformation with a birth prevalence of 7/1000. CHD may occur as Mendelian syndromic disorders or as isolated conditions. The latter represent the majority (~80%) of CHD cases. Recent technological advancements have allowed large-scale genome-wide characterization of copy number variants (CNVs), which have been proposed to contribute to the risk of sporadic CHD. This thesis presents a genome-wide CNV study involving 2256 sporadic, isolated CHD patients, 283 trio CHD families, and 1538 ancestry-matched controls that were typed on the Illumina 660W-Q SNP platform. This was followed by an extensive validation study using comparative genomic hybridization arrays, multiplex ligation-dependent probe amplification and quantitative-fluorescent PCR assays. A global enrichment of rare genic deletions was identified in CHD patients (OR = 1.8, P = 0.001), compared to controls. Rare deletions that are associated with CHD had higher gene content (P = 0.001) and higher haploinsufficiency scores (P = 0.03). Additionally, they were enriched with genes involved in the Wnt signalling pathway, known for its pivotal role in cardiac morphogenesis. Rare de novo CNVs were also identified in ~5% CHD trios; 91% of which occurred on the paternal, as opposed to the maternal chromosome (P = 0.01). They spanned previously known candidate loci as well as novel loci for CHD. Individual locus enrichments in cases vs. controls were identified for CNVs at chromosomes 1q21.1 and 15q11.2. A phenotype-specific effect was observed for the 1q21.1 CNVs, and GJA5 was identified as the causative gene for CHD in this locus. In conclusion, global rare genic deletions contribute ~4% of the population attributable risk of sporadic CHD. CNVs implicating 1q21.1, 15q11.2 and Wnt signalling genes are associated with CHD. Rare de novo CNVs identified in CHD trios exhibit a paternal origin bias possibly of relevance to the epidemiology of CHD. i Dedication Dedication for my parents Herman Soemedi and Dyan Chan ii Declaration and copyright Declaration and copyright No part of the work presented in this thesis has been previously submitted for a degree at Newcastle University or at any other institution. ©2012 - Rachel Soemedi All rights reserved. iii Table of Contents Table of Contents Abstract ............................................................................................................. i Dedication ......................................................................................................... ii Declaration and copyright .............................................................................. iii Table of Contents ............................................................................................ iv List of Figures ................................................................................................ viii List of Tables ................................................................................................... xi Acknowledgements ....................................................................................... xiii Statement of contributions ............................................................................ xv Abbreviations ................................................................................................ xvii 1 Introduction ................................................................................................ 1 1.1 Preface ................................................................................................ 2 1.2 Contribution of human genetic variation to complex traits ................... 2 1.3 Copy number variants ......................................................................... 4 1.3.1 Segmental duplications ............................................................. 6 1.3.2 Segmental duplication-mediated CNV formation ....................... 6 1.3.3 Other mechanisms for CNV formation ....................................... 9 1.3.4 Genome-wide CNV detection methods ................................... 13 1.3.5 Targeted CNV detection methods ........................................... 20 1.4 Congenital heart disease ................................................................... 23 1.4.1 Cardiac morphogenesis........................................................... 24 1.4.2 Types of congenital heart disease ........................................... 25 1.4.3 Genetic factors for CHD .......................................................... 30 1.4.4 Environmental factors for sporadic CHD ................................. 38 1.5 General aim ....................................................................................... 39 2 Materials and Methods ............................................................................ 40 2.1 Study Subjects ................................................................................... 41 2.1.1 Sample collections and inclusion criteria ................................. 41 2.1.2 French population cohort ......................................................... 41 2.1.3 WTCCC2 control cohort .......................................................... 41 2.2 CNV detection on SNP arrays ........................................................... 42 iv Table of Contents 2.2.1 QC procedures ........................................................................ 42 2.2.2 CNV calling algorithms ............................................................ 43 2.2.3 Contribution from collaborators in Statistical Genetics Group . 43 2.2.4 CNV analyses .......................................................................... 46 2.2.5 Database mining ..................................................................... 46 2.2.6 CNV validation ......................................................................... 50 2.3 Comparative Genomic Hybridization (CGH) ...................................... 51 2.3.1 DNA purification by ethanol precipitation ................................. 51 2.3.2 Fluorescent dUTP labelling ..................................................... 52 2.3.3 Purification of labelled DNAs ................................................... 53 2.3.4 Array hybridization ................................................................... 54 2.3.5 Array wash .............................................................................. 56 2.3.6 Scanning and analyses ........................................................... 56 2.4 Multiplex Ligation-dependent Probe Amplification (MLPA) ................ 57 2.4.1 MLPA design ........................................................................... 57 2.4.2 MLPA assay ............................................................................ 58 2.4.3 MLPA analyses ....................................................................... 59 2.5 Quantitative Fluorescence (QF) -PCR ............................................... 61 2.5.1 QF-PCR assay ........................................................................ 61 2.5.2 QF-PCR analyses ................................................................... 61 2.6 Statistical analyses ............................................................................ 63 2.6.1 CNV burden and gene-content analyses ................................. 63 2.6.2 Parental origin bias ascertainment .......................................... 63 2.6.3 Frequency of 1q21.1 rearrangements in cases vs. controls .... 64 2.6.4 Frequency of GJA5 duplications in cases versus controls ...... 64 2.6.5 Population attributable risk (PAR) ........................................... 64 3 Preliminary analyses to identify CHD patients with known causative CNVs ......................................................................................................... 65 3.1 Abstract ............................................................................................. 66 3.2 Aims .................................................................................................. 66 3.3 Results .............................................................................................. 67 3.3.1 Identification of patients with whole chromosomal aberrations 67 3.3.2 Identification of DiGeorge and Williams-Beuren CNVs ............ 71 v Table of Contents 3.4 Discussion ......................................................................................... 72 4 Global Rare Copy Number Variants Contribute to Sporadic Congenital Heart Disease ........................................................................................... 74 4.1 Abstract ............................................................................................. 75 4.2 Background ....................................................................................... 76 4.3 Aims .................................................................................................. 77 4.4 Results .............................................................................................. 78 4.4.1 CNV validation and inclusion criteria ....................................... 78 4.4.2 CNV burden in CHD cases and controls ................................