UNIVERSITY OF MANCHESTER Defining the key biological and genetic mechanisms involved in psoriasis A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health 2017 Helen F. Ray-Jones School of Biological Sciences Division of Musculoskeletal and Dermatological Sciences 1 2 Table of Contents 0. Introduction ................................................................................................................ 25 0.1 Disease prevalence ................................................................................................ 26 0.2 Phenotypes of psoriasis ......................................................................................... 26 0.3 Cellular basis .......................................................................................................... 29 0.4 Age of onset ........................................................................................................... 33 0.5 Environmental risk factors .................................................................................... 35 0.6 Quality of life ......................................................................................................... 36 0.7 Therapeutic treatments ........................................................................................ 37 0.7.1 Topical therapies ............................................................................................ 37 0.7.2 UV therapy ..................................................................................................... 37 0.7.3 Systemic treatments ...................................................................................... 38 0.7.4 Biologics ......................................................................................................... 38 0.7.4.1 Anti-TNFα therapies ................................................................................ 38 0.7.4.2 Anti- IL-12/IL-23 therapies ...................................................................... 39 0.7.4.3 Anti-IL-17 therapies ................................................................................ 39 0.7.4.4 Limitations of biologics ........................................................................... 40 0.8 Genetic risk factors ................................................................................................ 40 0.8.1 Linkage studies ............................................................................................... 41 0.8.2 Candidate gene studies .................................................................................. 41 0.8.3 Genome wide association studies ................................................................. 42 0.8.3.1 GWAS and fine mapping in psoriasis ...................................................... 44 0.8.3.2 Genetic overlap with psoriatic arthritis .................................................. 56 0.8.3.3 Genetics of late-onset psoriasis .............................................................. 56 0.9 Moving beyond GWAS ........................................................................................... 57 0.9.1 Interpreting GWAS data ................................................................................. 58 0.9.2 Functional annotation of GWAS loci .............................................................. 59 3 0.9.2.1 Importance of cell type .......................................................................... 60 0.9.2.2 Accessible chromatin .............................................................................. 61 0.9.2.3 Protein interactions ................................................................................ 62 0.9.2.4 Chromatin interactions .......................................................................... 64 0.9.2.5 Selected psoriasis risk loci for functional follow-up ............................... 69 0.10 Summary ............................................................................................................... 70 0.11 Overall aims and objectives .................................................................................. 71 0.12 Outline of thesis .................................................................................................... 71 1. A genome-wide association study of late-onset psoriasis ......................................... 73 1.1 Introduction .......................................................................................................... 74 1.2 Aims and objectives of Section 1 .......................................................................... 74 1.3 Methods ................................................................................................................ 75 1.3.1 Samples .......................................................................................................... 75 1.3.1.1 Cases ....................................................................................................... 75 1.3.1.2 Controls .................................................................................................. 76 1.3.2 Genotyping of the Manchester PsA cohort ................................................... 78 1.3.2.1 Illumina Infinium HTS Assay ................................................................... 78 1.3.2.2 GenomeStudio ........................................................................................ 80 1.3.2.3 Quality control of the Manchester PsA genotype data ......................... 80 1.3.3 Merging case-control datasets ...................................................................... 82 1.3.4 Imputation ..................................................................................................... 82 1.3.5 Association analysis ....................................................................................... 83 1.3.5.1 Frequentist test for association ............................................................. 83 1.3.5.2 Correction for multiple testing ............................................................... 84 1.3.5.3 Testing for independent signals in the MHC .......................................... 84 1.3.5.4 Annotation of results .............................................................................. 84 1.3.5.5 Post-analysis QC of novel signals ........................................................... 85 4 1.4 Results ................................................................................................................... 86 1.4.1 Samples .......................................................................................................... 86 1.4.2 Genotyping of the Manchester PsA cohort ................................................... 86 1.4.3 Merging case-control datasets ....................................................................... 87 1.4.4 Imputation ...................................................................................................... 89 1.4.5 Association analysis ........................................................................................ 89 1.4.5.1 Conditional analysis in the MHC ............................................................. 94 1.4.5.2 Overlap with other traits in GWAS datasets ........................................... 94 1.4.5.3 Replication of LOP signals ....................................................................... 95 1.4.5.4 Putative novel LOP loci ........................................................................... 99 1.5 Discussion ............................................................................................................108 1.5.1 Validation of known psoriasis loci ................................................................108 1.5.2 The 2q13 (IL1R1) locus .................................................................................109 1.5.3 Putative novel LOP loci .................................................................................110 1.5.4 Strengths and limitations .............................................................................113 1.5.5 Future work ..................................................................................................114 1.5.6 Conclusions ..................................................................................................115 2. Functional characterisation of psoriasis risk loci ......................................................117 2.1 Introduction .........................................................................................................118 2.2 Aims and objectives of Section 2 .........................................................................118 2.3 Methods ..............................................................................................................120 2.3.1 Methods for functional characterisation of individual risk loci ...................120 2.3.1.1 Bioinformatics .......................................................................................120 2.3.1.2 Chromatin Immunoprecipitation ..........................................................126 2.3.1.3 Chromosome conformation capture ....................................................139 2.3.1.4 Stimulation of HaCaT cells for ChIP and 3C in 9q31 .............................160 2.3.2 Methods for functional characterisation of multiple risk loci .....................165 5 2.3.2.1 HaCaT stimulation time-course and expression analysis ..................... 165 2.3.2.2 Capture Hi-C study...............................................................................