Investigation of a susceptibility locus for Psoriatic Arthritis

269 Pages·2016·22.65 MB·English

Investigation of a susceptibility
locus for Psoriatic Arthritis

A thesis submitted to The University of Manchester for
the degree of PhD
in the Faculty of Biology, Medicine and Health

2016

Ashley Budu-Aggrey

School of Biological Sciences
Table of Contents
List of Tables ................................................................................................................................ 7
List of Figures ............................................................................................................................. 10
List of Abbreviations .................................................................................................................. 13
Abstract ..................................................................................................................................... 18
Declaration ................................................................................................................................ 19
Copyright Statement .................................................................................................................. 19
Publications arising during this PhD ............................................................................................ 20
About the author ....................................................................................................................... 21
Acknowledgements .................................................................................................................... 22
Dedication ................................................................................................................................. 23
1. INTRODUCTION ................................................................................................................... 24
1.1 Background of psoriatic disease ..................................................................... 25
1.1.1 Epidemiology of psoriasis .................................................................................. 25
1.2 Clinical features ............................................................................................. 26
1.3 Psoriasis and arthritic disease ........................................................................ 27
1.3.1 Psoriatic Arthritis................................................................................................ 28
1.3.1.1 Epidemiology .............................................................................................. 28
1.3.1.2 Diagnosis .................................................................................................... 29
1.3.1.3 Pathogenesis .............................................................................................. 31
1.3.1.4 Treatment .................................................................................................. 33
1.3.1.5 Environmental risk factors ......................................................................... 36
1.4 Investigating the genetic basis of disease ....................................................... 38
1.4.1 Candidate-gene studies ..................................................................................... 38
1.4.2 Genome-wide association studies ..................................................................... 39
1.4.2.1 Linkage-disequilibrium and association studies ........................................ 40
1.4.3 Fine mapping studies ......................................................................................... 41

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1.4.4 Rare variant studies ........................................................................................... 42
1.4.4.1 Multiple-variant testing ............................................................................. 43
1.5 Genetics of Psoriatic Disease ......................................................................... 44
1.5.1 Genetic studies in psoriasis ................................................................................ 44
1.5.2 Genetic studies in PsA ........................................................................................ 47
1.5.2.1 HLA associations......................................................................................... 48
1.5.2.2 PsA GWAS Studies ...................................................................................... 50
1.6 Evidence of specific PsA risk loci..................................................................... 53
1.6.1 IL13 ..................................................................................................................... 54
1.6.2 HLA Alleles .......................................................................................................... 55
1.6.3 TNFAIP3 .............................................................................................................. 56
1.7 Genetic risk loci as therapeutic targets ........................................................... 57
1.8 Beyond GWAS studies ................................................................................... 59
1.8.1 Expression quantitative trait loci (eQTLs) .......................................................... 59
1.8.2 Functional variants ............................................................................................. 60
1.8.3 Transcribed regions ............................................................................................ 60
1.8.4 Chromatin structure and modification .............................................................. 61
1.8.5 Protein bound regions ....................................................................................... 61
1.9 Hypothesis .................................................................................................... 63
1.10 Objectives ..................................................................................................... 63
1.10.1 PsA Immunochip Study Objectives ................................................................ 63
1.10.2 Candidate-SNP study objectives .................................................................... 63
1.10.3 PsA rare variant study objectives ................................................................... 64
2. METHODS AND MATERIALS ................................................................................................. 65
2.1 Psoriatic Arthritis Immunochip study ............................................................. 66
2.1.1 PsA case and control samples ............................................................................ 66
2.1.2 Genotyping overview ......................................................................................... 67
2.1.3 Genotyping protocol .......................................................................................... 68

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2.1.3.1 Day one of the Illumina Infinium® HD Assay Ultra Protocol ...................... 68
2.1.3.2 Day two of the Illumina Infinium® HD Assay Ultra Protocol ...................... 69
2.1.3.3 Day three of the Illumina Infinium® HD Assay Ultra Protocol ................... 72
2.1.4 Data aggregation ................................................................................................ 76
2.1.5 Statistical analysis .............................................................................................. 76
2.1.5.1 Statistical quality control ........................................................................... 76
2.1.5.2 Association testing ..................................................................................... 78
2.1.6 Bioinformatic analysis ........................................................................................ 79
2.1.6.1 SNP selection .............................................................................................. 80
2.1.6.2 Use of eQTL databases ............................................................................... 80
2.1.7 Cell specific eQTL analysis .................................................................................. 95
2.2 PsA candidate-SNP study ............................................................................... 97
2.2.1 PsA case and control samples ............................................................................ 97
2.2.2 SNP genotyping .................................................................................................. 99
2.2.3 Statistical analysis ............................................................................................ 101
2.2.4 Functional annotation ...................................................................................... 103
2.3 Rare variant study ....................................................................................... 105
2.3.1 PsA case and control samples .......................................................................... 105
2.3.2 Genotyping protocol ........................................................................................ 106
2.3.3 Statistical analysis ............................................................................................ 106
3. RESULTS ............................................................................................................................ 110
3.1 PsA Immunochip Study ................................................................................ 111
3.1.1 Association testing ........................................................................................... 113
3.1.2 Bioinformatic analysis ...................................................................................... 115
3.1.2.1 Associated SNP set ................................................................................... 115
3.1.2.2 Identifying candidate genes ..................................................................... 116
3.1.2.3 Identifying candidate SNPs ...................................................................... 124
3.1.3 Cell specific analysis ......................................................................................... 132

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3.1.3.1 Identifying critical cell types for PsA ........................................................ 132
3.1.3.2 Cell type-specific eQTL study ................................................................... 135
3.1.3.3 Summary of PsA Immunochip study ........................................................ 137
3.2 PsA Candidate SNP study ............................................................................. 138
3.2.1 Statistical analysis ............................................................................................ 138
3.2.1.1 Investigating independent effects at the IL23R locus .............................. 142
3.2.1.2 Summary of PsA candidate-SNP study ..................................................... 147
3.3 PsA rare variant study ................................................................................. 147
3.3.1 Sample QC ........................................................................................................ 147
3.3.2 SNP QC ............................................................................................................. 148
3.3.3 Association testing ........................................................................................... 152
3.3.3.1 Common variants ..................................................................................... 152
3.3.4 Rare and low-frequency variants ..................................................................... 160
3.3.4.1 Single-variant analysis .............................................................................. 160
3.3.4.2 Multiple-variant analysis .......................................................................... 165
3.3.5 Investigating the IFIH1 risk locus ..................................................................... 170
3.3.5.1 Conditional logistic regression ................................................................. 170
3.3.5.2 Haplotype analysis ................................................................................... 172
3.3.5.3 Summary of PsA rare variant study ......................................................... 173
4. DISCUSSION ...................................................................................................................... 174
4.1 Summary..................................................................................................... 175
4.2 PsA Immunochip study ................................................................................ 176
4.2.1 Strengths and weaknesses of the Immunochip study ..................................... 176
4.2.2 PsA-specific association on chromosome 5q31 ............................................... 178
4.2.3 Identifying candidate causal genes - bioinformatic evidence .......................... 179
4.2.4 Identifying candidate causal genes - cell type-specific eQTL analysis ............. 181
4.2.5 Identifying candidate causal variants .............................................................. 182
4.2.6 Implications of the PsA Immunochip study ..................................................... 183

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4.3 PsA candidate SNP study ............................................................................. 184
4.3.1 Strengths and weaknesses of the candidate SNP study .................................. 184
4.3.2 Significant associations .................................................................................... 185
4.3.3 A distinct PsA association at the IL23R locus ................................................... 186
4.3.4 Functional annotation of the IL23R locus ........................................................ 188
4.3.5 Implications of the candidate SNP study ......................................................... 189
4.4 Rare variant analysis in PsA ......................................................................... 190
4.4.1 Strengths and weaknesses of the rare variant study ....................................... 190
4.4.2 Associations with established loci.................................................................... 193
4.4.3 Association with IFIH1 ...................................................................................... 193
4.4.4 Implications of the rare variant PsA study ....................................................... 194
4.5 Future work ................................................................................................ 195
4.5.1 Functional validation of causal variants ........................................................... 195
4.5.2 Functional validation of causal genes .............................................................. 197
4.5.3 Identifying additional causal variants and genes ............................................. 198
4.6 Conclusions ................................................................................................. 199
5. APPENDIX ......................................................................................................................... 201
Appendix A PsA Immunochip study ..................................................................... 202
Appendix B Phenotypic Cell Type Specificity software manual ............................. 210
Appendix C Candidate-SNP study association results ........................................... 214
Appendix D Rare variant analysis results ............................................................. 218
Appendix E Publications arising during the PhD ................................................... 222
6. REFERENCES ...................................................................................................................... 254

Word count of main text: 61,929

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List of Tables
Table 1 - The Moll and Wright 1973 classification criteria for psoriatic arthritis .................. 30
Table 2 - The CASPAR (Classification of Psoriatic Arthritis) criteria for the diagnosis of
psoriatic arthritis.. .................................................................................................................. 30
Table 3 – Patient response from trials investigating the efficacy of anti-TNFα treatments for
PsA.......................................................................................................................................... 35
Table 5 - Genome-wide significant assoications currently reported for PsA. ....................... 48
Table 6 - Summary of samples genotyped by centre ............................................................ 67
Table 7 - RegulomeDB scores are assigned to SNPs based on evidence of regulatory
function .................................................................................................................................. 82
Table 8 – eQTL datasets included in the RegulomeDB database........................................... 83
Table 9 - Datasets used to source gene expression data for Genevar database ................... 85
Table 10 - Datasets used to source gene expression data for GTEx database ...................... 87
Table 11 - Datasets used to source gene expression data for The Pritchard Lab eQTL
Browser .................................................................................................................................. 90
Table 12 - Summary of datasets queried in eQTL databases ................................................. 94
Table 13 - Variants selected to be genotyped in PsA replication cohorts ............................. 98
Table 14 - PsA replication samples genotyped as part of the PsA candidate SNP study ....... 99
Table 15 - Genome markers that are unique and common between the HumanExome chip
and the HumanCoreExome chip .......................................................................................... 105
Table 16 - PsA cases and healthy controls genotyped as part of the PsA rare variant study
............................................................................................................................................. 105
Table 17 - Example of a SNP excluded due to bias in genotyping ....................................... 107
Table 18 - Association statistics for novel risk locus at chromosome 5q31 ........................ 113
Table 20- Results obtained from RegulomeDB eQTL database ........................................... 117
Table 21 - eQTLs identified from the Genvar eQTL database .............................................. 118
Table 22 - eQTLs identified from the GTEx eQTL browser ................................................... 119
Table 23 - eQTLs retrieved from the GHS_Express database .............................................. 121
Table 24 - eQTLs retrieved from The Pritchard Lab eQTL Browser ..................................... 123
Table 25 - Evidence of cell type-specific effects from various eQTL studies ....................... 124
Table 26 - Summary of results obtained from RebulomeDB database ............................... 125
Table 27 - Functional variants identified from RegulomeDB ............................................... 125
Table 28 - Functional variants identified from the Assimilator tool .................................... 126

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Table 29 - Cell-type specificity scores for overlap of PsA associated variants with H3K4me3
chromatin mark peaks in CD8+ memory primary T-cells. bp; base position.. ...................... 133
Table 30 - Enrichment of 20 PsA associated variants at H3K4me3 chromatin marks in 34 cell
and tissue types ................................................................................................................... 134
Table 31 - Post-QC PsA replication samples genotyped as part of the PsA candidate SNP
study ..................................................................................................................................... 139
Table 32 - Association statistics of the candidate SNPs genotyped in PsA replication cohorts.
............................................................................................................................................. 140
Table 33 - Summary statistics for Immunochip and meta-analysis of candidate SNPs. ...... 141
Table 34 - Functional annotation of IL23R credible SNP sets using data from the Roadmap
Epigenomics Project. ............................................................................................................ 145
Table 35 - Summary of SNP QC, call rate threshold = 0.98. ................................................. 150
Table 36 - Summary of SNP QC, call rate threshold = 0.99. ................................................. 151
Table 37 - Summary of SNP QC, call rate threshold = 1.0. ................................................... 151
Table 38 - Calculated genomic inflation factor for rare and common variants ................... 152
Table 39 - Association statistics of common variants identified at previously reported GWAS
loci. SNP call rate threshold = 1.0.. ...................................................................................... 158
Table 40 - Association statistics of most strongly associated low-frequency and rare variants
identified by performing single-point analysis (P < 1.0x10-4, SNP call rate = 1.0). .............. 161
Table 41 - Strongest associations identified from multiple-variant testing (P<1.0x10-03, SNP
call rate > 0.99) .................................................................................................................... 166
Table 42 - Strongest associations identified from multiple-variant testing (P<1.0x10-03, SNP
call rate = 1.0) ...................................................................................................................... 166
Table 43 - Summary statistics of IFIH1 variants from single-variant analysis ...................... 168
Table 44 – Haplotypes defined with IFIH1 variants, rs35667974 and rs984971 ................. 173
Table 45 - Benefits and limitations of GWAS studies using arrays available at the time
compared with Immunochip studies ................................................................................... 177
Table 46- Initial quality control of Immunochip study data using GenomeStudio Genotyping
software ............................................................................................................................... 202
Table 47 - Quality control of Immunochip study samples ................................................... 203
Table 48 - Results retrieved from Genevar eQTL database for all genes ............................ 204
Table 49 - ENCODE tracks queried by the Assimilator tool ................................................. 207
Table 50 - Summary statistics for association testing with Cretan PsA cases and controls. 214

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Table 51 - Summary statistics for association testing with Spanish PsA cases and controls.
............................................................................................................................................. 215
Table 52 - Summary statistics for association testing with German PsA cases and controls.
............................................................................................................................................. 215
Table 53 - Summary statistics for association testing with UK PsA cases and controls....... 216
Table 54 - Association statistics for rare and low-frequency variants (call rate > 0.98, P <
1.0x10-3). .............................................................................................................................. 219
Table 55 - Association statistics for rare and low-frequency variants (call rate > 0.99, P <
1.0x10-3). .............................................................................................................................. 220

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List of Figures
Figure 1 - Clinical presentations of psoriasis .......................................................................... 27
Figure 2 - Patient with clinical features of psoriasis and PsA ................................................ 29
Figure 3 – Main points of inflammation in PsA ...................................................................... 32
Figure 4 - A Genome-Wide Association Scan ......................................................................... 40
Figure 5 - Connection between genetic risk loci and therapeutic targets ............................. 58
Figure 6 - Overview of Immunochip Assay workflow ............................................................ 68
Figure 7 - Hybridisation of sample DNA to Infinium HD BeadChips ...................................... 71
Figure 8 - Scheme for dispensing DNA samples from 96 well plates onto 1x12 Infinium HD
BeadChips .............................................................................................................................. 71
Figure 9 - Washing of non-hybridised and non-specifically hybridised DNA from Infinium HD
BeadChips .............................................................................................................................. 72
Figure 10 - Single-base extension of oligo-probes on Infinium HD BeadChips ...................... 73
Figure 11 - Protocol followed for single-base extension of oligo-probes on Infinium HD
BeadChips .............................................................................................................................. 74
Figure 12 - Protocol followed for staining of the Infinium HD BeadChips ............................. 75
Figure 13 - Summary of bioinformatic analysis to investigate the regulatory function of the
associated region ................................................................................................................... 79
Figure 14 - Use of RegulomeDB database to provide evidence of functional variants ......... 84
Figure 15 - GENe Expression VARiation (Genevar) eQTL database to identify eQTLs ........... 86
Figure 16 - Use of Genome-Tissue Expression eQTL (GTEx) Browser to identify eQTLs ....... 88
Figure 17 - Use of GHS_Express database to identify eQTLs ................................................. 89
Figure 18 - Use of The Pritchard Lab eQTL Browser to identify eQTLs .................................. 91
Figure 19 - Use of Assimilator to identify SNPs with evidence of regulatory function .......... 92
Figure 20 - Identifying relevant cell types for disease ........................................................... 96
Figure 21 - TaqMan® genotyping assay ............................................................................... 100
Figure 22 - Example of good genotype clustering with the TaqMan® Genotyper Software102
Figure 23 - HaploReg version 4.1. ........................................................................................ 104
Figure 24 - Genetic and epigenetic fine mapping data portal. ............................................ 104
Figure 25 - Stages of methods applied to the Immunochip project .................................... 112
Figure 26 – Results from PsA Immunochip study ................................................................ 114
Figure 27 - Correlation between expression of P4HA2 and rs3846728 ............................... 120
Figure 28 - Track displaying eQTLs retrieved from The Pritchard Lab eQTL Browser ......... 122

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