Systematic analysis of genomic copy number variations in inflammatory bowel diseases Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Christian-Albrechts-Universität zu Kiel vorgelegt von Hamidreza Saadati Kiel, Dezember 2016 Referent/in: Prof. Dr. Andre Franke Koreferent/in: Prof. Dr. Manuela Dittmar Tag der mündlichen Prüfung: 15.12.2016 gez. Prof. Dr. Natascha Oppelt, Dekanin Table of contents List of figures.......................................................................................................................v List of Tables......................................................................................................................vi 1 Introduction..........................................................................................................................1 1.1 Inflammatory bowel diseases (IBD)..........................................................................................1 1.1.1 Epidemiology of IBD......................................................................................................5 1.1.2 Genetic architecture of IBD............................................................................................6 1.1.2.1. Gene discovery approaches........................................................................6 1.1.2.2. Identified genes and implicated pathways...................................................9 1.2 Copy number variations (CNVs).............................................................................................16 1.2.1 Discovery and mapping of CNVs.................................................................................19 1.2.2 Mechanisms of evolving CNVs....................................................................................24 1.2.3 Functional consequences of CNVs..............................................................................29 1.2.4 CNVs and diseases.....................................................................................................31 1.3 Monozygotic (MZ) twins...........................................................................................................36 1.3.1 Discordant MZ twins in disease studies.......................................................................38 1.3.2 Genetic differences in MZ twins: somatic mosaicism..................................................39 1.3.3 MZ twins in IBD............................................................................................................42 1.4 Aims of this study.....................................................................................................................44 2 Methods.............................................................................................................................45 2.1 Twin sample recruitment........................................................................................................45 2.2 Twin sample preparations......................................................................................................46 2.2.1 DNA extraction from blood and biopsy samples.........................................................46 2.2.2 Twin sample combinations..........................................................................................48 2.3 Array-CGH experiments..........................................................................................................49 2.3.1 Array-CGH workflow....................................................................................................50 2.4 UC case-control cohorts..........................................................................................................53 2.4.1 Patient recruitment and ethics.....................................................................................55 2.5 CNV calling of Affy6.0 datasets...............................................................................................56 2.6 Screening for rare CNVs in Affy6.0 datasets...........................................................................59 2.7 Association analysis for common CNVs..................................................................................60 2.8 Technical validation and replication of predicted CNVs..........................................................61 2.8.1 TaqMan® copy number analysis................................................................................61 2.8.2 Copy number data analysis of TaqMan®CNV assays................................................64 3 Results...............................................................................................................................66 3.1 CNV identification in MZ twins................................................................................................66 3.1.1 Primary array-CGH results..........................................................................................66 3.1.2 Technical validations of the predicted CNVs...............................................................67 3.1.3 Accuracy testing of platform for twin CNV analysis.....................................................68 3.2 CNV analysis in UC case-control panels.................................................................................72 3.2.1 Screening in German discovery panel.........................................................................73 3.2.2 Visual inspection of initial CNV regions.......................................................................74 3.2.3 Relevant common CNV regions in German discovery panel.......................................75 3.2.4 Following in independent replication panels................................................................76 3.2.5 Evaluation of the relevant CNVs in in-silico controls.......................................................78 3.2.6 Technical validations of the three relevant CNVs........................................................80 4 Discussion.........................................................................................................................86 4.1 Rare CNVs overrepresented in UC cases.............................................................................87 4.2 No confirmed CNVs in MZ twins discordant for IBD..............................................................90 4.3 Methodological pitfalls...........................................................................................................93 4.3.1 The problem of wave artifacts in twin array-CGH analysis..........................................93 4.3.2 Deficient probe coverage and difficulties in genotyping multi-allelic CNVs.................94 4.3.3 Low consistency of CNV detection algorithms.............................................................96 4.4 Probable sources of discordance in IBD MZ twins.................................................................98 4.4.1 Epigenetic factors........................................................................................................98 4.4.2 Gut microbiota............................................................................................................100 4.4.3 Other environmental factors.......................................................................................102 4.5 Missing heritability and CNVs................................................................................................106 4.5.1 Inflated (phantom) heritability for IBD?......................................................................106 4.5.2 Where (how) to find the probable missing variants for IBD........................................108 4.6 Concluding remarks...............................................................................................................109 5 References......................................................................................................................111 6 Supplementary material..................................................................................................123 7 Summary.........................................................................................................................174 8 Zusammenfassung..........................................................................................................176 Curriculum Vitae.............................................................................................................178 Declaration.....................................................................................................................179 Acknowledgment............................................................................................................180 v List of Figures Fig. 1.1 Inverse relations between incidence of infectious diseases and the immune disorders................1 Fig. 1.2 Upper and lower human gastrointestinal tract and general structure of the gut wall.....................2 Fig. 1.3 Histological hallmarks of IBD.........................................................................................................4 Fig. 1.4 Features of disease-associated genetic variants...........................................................................8 Fig. 1.5 Key features of the intestinal immune system.............................................................................14 Fig. 1.6 Biological processes implicated by IBD loci.................................................................................15 Fig. 1.7 Major categories of genetic variants............................................................................................16