The interleukin 1 gene family in systemic juvenile idiopathic arthritis

413 Pages·2011·13.14 MB·English

The Interleukin 1 Gene
Family in Systemic Juvenile
Idiopathic Arthritis

Carmel Joanna Winstanley Stock

A thesis submitted for the degree of Doctor of Philosophy to
University College London

2011

Centre for Paediatric and Adolescent Rheumatology
Department of Immunology and Molecular Pathology
Division of Infection and Immunity
Windeyer Institute of Medical Sciences
University College London
46 Cleveland Street
London
W1T 4JF
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Abstract
Patients with systemic Juvenile Idiopathic Arthritis (sJIA) have elevated serum levels of
inflammatory cytokines. Treatment with interleukin-1 (IL-1) receptor antagonist (Anakinra)
shows remarkable improvement in some sJIA patients. The hypothesis of this thesis is that
genetic variations in IL-1 family genes contribute to disease pathogenesis.

To investigate this, a two-stage case-control association study of 20 candidate genes was
performed. Selected tagging SNPs were tested for association in 130 sJIA patients and 146
controls in stage-1 of the study. SNPs at significantly different frequencies in the cohorts
were genotyped in an additional 105 sJIA patients and 184 controls, and stratified meta-
analysis of the two-stage data performed. Analysis was also performed with 4,671 controls
from the Wellcome Trust Case Control Consortium (WTCCC).

No associations were found with caspase-1, cryopyrin, or IL-18. Significant disease
associations were identified with SNPs in the ligand IL1A, the receptor antagonist IL1RN, and
a two-SNP haplotype in the IL-18 antagonist IL18BP. Associations were also identified in the
decoy receptor IL1R2, and the co-receptor IL1RAP, although these were not confirmed when
re-analysed with the WTCCC controls. Transient transfection assays with haplotype
constructs, performed by Dr Wen, showed that the IL18BP haplotype affected gene
transcription levels in vitro. This effect was not however reproduced using PBMCs from
healthy individuals. Allele specific binding to one of the haplotype SNPs was predicted in
silico, but no evidence for this was seen in EMSA experiments. Further functional studies are
required to corroborate involvement of this haplotype in disease.

In summary, this study has identified genetic associations for susceptibility to sJIA with a
number of IL1 family members. These results indicate that there may be aberrant control of
IL-1 activity in patients with sJIA. Further work is required to determine how these
associated SNPs affect IL-1 activity, and thereby the inflammatory response in sJIA.

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Acknowledgements

I would like to thank Professor Woo for giving me the opportunity to work in her group, and
her support and supervision throughout my project.

I would also like to thank Dr Mark Fife for his continued supervision, and Professor Cathryn
Lewis for advice and guidance on the statistical analysis.

I am very grateful to all of the members of the ‘Woo crew’ who I have worked with,
especially Dr Jane Samuel, Miss Emma Ogilvie, Dr Ebun Omyonmi, Dr Dongling Zheng,
and Dr Aiqing Wen who performed the transcription study.

I would like to thank Professor David Isenberg and the Oliver Bird Rheumatism Program for
giving me the opportunity to do this PhD.

I would also like to thank all of the patients and healthy individuals who kindly donated DNA
and blood samples, and the members of BSPAR who contributed to the DNA repository,
without which this project would not have been possible.

Thank you to my parents for their support and understanding.

A huge thanks to my fellow Olli Birdies, who were always there to share problems and
successes, as well as a few drinks.

I would also like to thank my partner Benit, whose support helped me to get to the end.
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Table of Contents
Abstract .......................................................................................................................... 2
Acknowledgements ......................................................................................................... 3
Table of Contents ............................................................................................................ 4
Table of Tables ..............................................................................................................10
Table of Figures .............................................................................................................12
Abbreviations .................................................................................................................14
Glossary .........................................................................................................................17
1. Diseases .................................................................................................................. 17
2. Disease features ....................................................................................................... 19
3. Measures of disease activity .................................................................................... 21
4. Medications ............................................................................................................. 22
5. Cell lines ................................................................................................................. 23
6. Somatic cells ........................................................................................................... 23
7. Immunomodulatory molecules ................................................................................. 25
8. Nomenclature of the genes investigated in this study ............................................... 28
1. Introduction ............................................................................................................30
1.1. Juvenile Idiopathic Arthritis ................................................................................. 30
1.2. Systemic Juvenile Idiopathic Arthritis .................................................................. 30
1.2.1. Complications ................................................................................................ 31
1.2.2. Diagnosis ....................................................................................................... 34
1.2.3. Treatment ...................................................................................................... 34
1.3. Inflammation and Cytokines ................................................................................ 40
1.4. Altered Cytokine Profile in sJIA .......................................................................... 42
1.5. Genetic Associations ........................................................................................... 45
1.5.1. Major Histo-compatibility Complex ............................................................... 46
1.5.2. Non-MHC Genes ........................................................................................... 46
1.6. sJIA is distinct from other JIA subtypes ............................................................... 49
1.7. Interleukin 1 Gene Family ................................................................................... 50
1.7.1. Activity .......................................................................................................... 50
1.7.2. IL-1 Ligands .................................................................................................. 50
1.7.2.1. IL-1α ...................................................................................................... 51
1.7.2.2. IL-1β ...................................................................................................... 51
1.7.3. IL-1Ra ........................................................................................................... 52
1.7.4. IL-1 Receptor Complex .................................................................................. 52
1.7.4.1. IL-1 R1 ................................................................................................... 52
1.7.4.2. IL-1R2 .................................................................................................... 54
1.7.4.3. IL-1RAcP ............................................................................................... 54
1.7.5. IL-18.............................................................................................................. 56
1.7.5.1. IL-18 Binding Protein ............................................................................. 56
1.7.5.2. IL-18 Receptor Complex ......................................................................... 57
1.7.6. Other IL-1 Family Members .......................................................................... 58
1.7.6.1. Ligands ................................................................................................... 58
1.7.6.2. Receptors ................................................................................................ 59
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1.7.7. IL-1 associated proteins ................................................................................. 60
1.7.7.1. Caspase-1 ................................................................................................... 61
1.7.7.2. Cryopyrin ................................................................................................... 61
1.7.8. IL-1 Family Gene Organisation ...................................................................... 62
1.8. IL-1 in disease ..................................................................................................... 62
1.9. IL-1 Gene Family Disease Associations ............................................................... 65
1.10. IL-1 in sJIA ......................................................................................................... 67
1.11. Hypothesis of the project ..................................................................................... 68
1.12. Aims of the project .............................................................................................. 68
2. Materials ................................................................................................................70
2.1. Patient and Control Samples ................................................................................ 70
2.1.1. Healthy controls ............................................................................................. 70
2.1.2. Patients .......................................................................................................... 70
2.2. Laboratory reagents ............................................................................................. 71
2.2.1. Chemicals ...................................................................................................... 71
2.2.2. Buffers ........................................................................................................... 73
2.2.3. Gels ............................................................................................................... 74
2.2.4. DNA extraction .............................................................................................. 75
2.2.5. PCR ............................................................................................................... 75
2.2.6. DNA purification ........................................................................................... 75
2.2.7. Cell culture .................................................................................................... 76
2.2.8. PBMC isolation ............................................................................................. 76
2.2.9. Bacterial culture ............................................................................................. 76
2.2.10. Cloning ...................................................................................................... 77
2.2.11. RNA quantification .................................................................................... 79
2.2.12. ELISA ........................................................................................................ 79
2.2.13. EMSA ........................................................................................................ 80
3. Methods .................................................................................................................82
3.1. Association Study ................................................................................................ 82
3.1.1. Introduction ................................................................................................... 82
3.1.1.1. Tagging SNPs ......................................................................................... 82
3.1.1.2. Two-stage study design ........................................................................... 85
3.1.2. Stage-1 ........................................................................................................... 86
3.1.2.1. tSNP Selection ........................................................................................ 86
3.1.2.2. Genotyping ............................................................................................. 97
3.1.2.2.1. Genotype calling ................................................................................ 97
3.1.2.2.2. Golden Gate genotyping pilot study ................................................. 101
3.1.2.3. Data Quality Controls ........................................................................... 101
3.1.2.4. Association Analysis ............................................................................. 104
3.1.3. Stage-2 ......................................................................................................... 105
3.1.3.1. Genotyping ........................................................................................... 105
3.1.3.2. Data Quality Controls ........................................................................... 105
3.1.3.3. Association Analysis ............................................................................. 106
3.1.4. WTCCC control cohort analysis ................................................................... 107
3.1.5. Identification of additional tagged SNPs ...................................................... 108
3.1.6. Validation of the tagging SNP selection results ................................................ 108
3.2. Sequence feature prediction .................................................................................... 109
3.2.1. Comparative Genomics ................................................................................ 110
3.2.2. Transcription Factor Binding prediction ............................................................... 113
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3.3. General experimental protocols .......................................................................... 113
3.3.1. DNA extraction ............................................................................................ 113
3.3.1.1. From blood ........................................................................................... 113
3.3.1.2. From saliva ........................................................................................... 114
3.3.1.3. Nucleic acid Quantification ................................................................... 114
3.3.2. Polymerase Chain Reaction (PCR) ............................................................... 114
3.3.2.1. Primer Design ....................................................................................... 114
3.3.2.2. PCR reaction ......................................................................................... 115
3.3.2.3. PCR cycling .......................................................................................... 115
3.3.2.4. Optimisation ......................................................................................... 116
3.3.2.5. PCR product visualisation ..................................................................... 116
3.3.2.6. PCR Product Purification ...................................................................... 116
3.3.2.7. PCR Product Gel Extraction .................................................................. 116
3.3.3. Genotyping of Healthy Controls ................................................................... 118
3.3.4. Cloning ........................................................................................................ 118
3.3.4.1. Generation of insert .............................................................................. 118
3.3.4.2. PCR Product Ligation into Plasmid ....................................................... 118
3.3.4.3. Plasmid Transformation into Competent Cells ...................................... 119
3.3.4.4. Plasmid amplification ........................................................................... 119
3.3.4.5. Colony PCR .......................................................................................... 119
3.3.4.6. Small scale plasmid preparation ‘Miniprep’ .......................................... 119
3.3.4.7. Sequencing ........................................................................................... 120
3.3.5. Cell culture .................................................................................................. 120
3.3.5.1. Peripheral Blood Mononuclear Cells (PBMCs) ..................................... 120
3.3.5.2. THP-1 Cells .......................................................................................... 120
3.3.5.3. Cell counting ........................................................................................ 120
3.3.6. PBMC isolation ........................................................................................... 121
3.3.7. Enzyme Linked Immunosorbant Assay (ELISA) .......................................... 121
3.3.7.1. Sandwich ELISA .................................................................................. 123
3.3.7.2. ELISA Analysis .................................................................................... 123
3.3.7.2.1. Standard Curve ................................................................................ 123
3.3.7.2.2. Statistical analysis ............................................................................ 123
3.3.8. RNA based protocols ................................................................................... 124
3.3.8.1. Total RNA extraction ............................................................................ 124
3.3.8.2. Reverse Transcription ........................................................................... 124
3.3.8.3. Quantitative RT-PCR ............................................................................ 125
3.3.8.3.1. Analysis ........................................................................................... 126
3.3.9. Electrophoretic Mobility Shift Assay (EMSA) ............................................. 126
3.3.9.1. Nuclear extraction ................................................................................. 126
3.3.9.2. Oligonucleotide annealing ..................................................................... 128
3.3.9.3. Probe amplification ............................................................................... 128
3.3.9.4. Probe labelling ...................................................................................... 128
3.3.9.5. Protein binding ..................................................................................... 129
3.3.9.6. Visualising DNA-protein complexes ..................................................... 130
3.4. IL18BP candidate region ................................................................................... 130
3.4.1. Investigation of expression levels according to haplotype ............................ 130
3.4.1.1. Haplotype determination ....................................................................... 130
3.4.1.1.1. Haplotype determination of uncertain phase ..................................... 130
3.4.1.2. PBMC stimulation ................................................................................ 131
3.4.1.3. Protein expression levels ....................................................................... 133
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3.4.1.3.1. Optimisation of cell concentration ................................................... 133
3.4.1.3.2. Expression comparison between haplotypes ..................................... 134
3.4.1.4. RNA expression levels .......................................................................... 134
3.4.1.4.1. Validation of housekeeping genes .................................................... 134
3.4.1.4.2. Expression comparison between haplotypes ..................................... 135
3.4.2. Transcription factor binding ......................................................................... 135
3.4.2.1. Probe design ............................................................................................. 135
3.4.2.2. Short probe EMSAs .................................................................................. 135
3.4.2.3. Long probe EMSAs .................................................................................. 137
3.5. Association analysis of the susceptibility alleles according to response to IL-1
blockade ........................................................................................................................ 137
4. Results ................................................................................................................. 140
4.1. Association Study Quality Control ..................................................................... 140
4.1.1. Power Calculation ........................................................................................ 140
4.1.2. Golden Gate genotyping pilot study ............................................................. 140
4.1.3. Stage-1 ............................................................................................................ 140
4.1.4. Stage-2 ......................................................................................................... 143
4.1.5. Validation of the tagging SNP selection results ................................................ 149
4.1.5.3. HapMap3 compared to WTCCC2 ............................................................. 158
4.2. IL18BP candidate region ................................................................................... 163
4.2.1. Association Study ........................................................................................ 163
4.2.1.1. Tagging SNP selection .......................................................................... 163
4.2.1.2. Stage-1.................................................................................................. 166
4.2.1.3. Stage-2.................................................................................................. 170
4.2.1.3.1. Meta-analysis................................................................................... 170
4.2.1.4. WTCCC control cohort analysis ............................................................ 170
4.2.1.5. Associated SNPs ................................................................................... 173
4.2.1.5.1. Additional captured SNPs................................................................... 173
4.2.1.5.2. Associated SNP positions ................................................................... 177
4.2.1.6. Summary .............................................................................................. 177
4.2.1.7. Discussion ............................................................................................ 180
4.2.2. Comparative Genomics .................................................................................... 182
4.2.2.1. Discussion................................................................................................. 182
4.2.3. Investigation of expression levels according to haplotype ............................ 187
4.2.3.1. Haplotype determination ....................................................................... 187
4.2.3.1.1. Haplotype determination of uncertain phase ..................................... 187
4.2.3.2. Protein expression levels ....................................................................... 187
4.2.3.2.1. Optimisation of cell concentration ................................................... 187
4.2.3.2.2. IL-18BP gene expression comparison according to haplotype .......... 190
4.2.3.3. RNA expression levels .......................................................................... 193
4.2.3.3.1. Validation of house keeping genes ................................................... 193
4.2.3.3.2. IL-18BP gene expression comparison according to haplotype .......... 197
4.2.3.4. Summary .................................................................................................. 201
4.2.3.5. Discussion................................................................................................. 201
4.2.4. Transcription Factor Binding ........................................................................... 203
4.2.4.1. Binding Prediction .................................................................................... 203
4.2.4.2. EMSA ....................................................................................................... 203
4.2.4.2.1. Short probe EMSAs ........................................................................... 203
4.2.4.2.2. Long probe EMSAs ............................................................................ 206
4.2.4.3. Summary .................................................................................................. 206
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4.2.4.4. Discussion................................................................................................. 206
4.3. IL1RAP candidate region................................................................................... 211
4.3.1. Association Study ........................................................................................ 211
4.3.1.1. tSNP selection ...................................................................................... 211
4.3.1.2. Stage-1 ...................................................................................................... 214
4.3.1.3. Stage- 2 ..................................................................................................... 231
4.3.1.3.1. Stratified analysis ............................................................................ 235
4.3.1.4. WTCCC control cohort analysis ................................................................ 237
4.3.1.4. Associated SNPs ................................................................................... 237
4.3.1.4.1. Additional captured SNPs ................................................................ 237
4.3.1.4.2. Associated SNP positions ................................................................ 237
4.3.1.5. Summary .................................................................................................. 240
4.3.1.6. Discussion................................................................................................. 240
4.4. IL1 ligand cluster candidate region .................................................................... 244
4.4.1. Association study ......................................................................................... 244
4.4.1.1. tSNP selection ...................................................................................... 244
4.4.1.2. Stage-1.................................................................................................. 247
4.4.1.3. Stage-2.................................................................................................. 262
4.4.1.3.1. Stratified analysis ................................................................................ 262
4.4.1.4. WTCCC control cohort analysis ............................................................ 268
4.4.1.5. Associated SNPs ................................................................................... 270
4.4.1.5.1. Additional captured SNPs ................................................................ 270
4.4.1.5.2. Associated SNP positions ................................................................... 272
4.4.1.6. Summary .................................................................................................. 272
4.4.1.7. Discussion................................................................................................. 275
4.5. IL1 receptor cluster candidate region ...................................................................... 279
4.5.1. Association Study ............................................................................................ 279
4.5.1.1. Tagging SNP selection .............................................................................. 279
4.5.1.2. Stage-1 ...................................................................................................... 281
4.5.1.3.1. Stratified analysis ............................................................................... 302
4.5.1.4. WTCCC control cohort analysis ................................................................ 302
4.5.1.5. Associated SNPs ....................................................................................... 302
4.5.1.5.1. Additional captured SNPs................................................................... 306
4.5.1.5.2. Associated SNP positions ................................................................... 306
4.5.1.6. Summary .................................................................................................. 306
4.5.1.7. Discussion................................................................................................. 309
4.6. IL18 candidate region ........................................................................................ 312
4.6.1. Association Study ........................................................................................ 312
4.6.1.1. tSNP selection ...................................................................................... 312
4.6.1.2. Stage-1.................................................................................................. 315
4.6.2. Summary ..................................................................................................... 315
4.6.3. Discussion ................................................................................................... 315
4.7. CASP1 candidate region .................................................................................... 320
4.7.1. Association Study ........................................................................................ 320
4.7.1.1. Tagging SNP selection .......................................................................... 320
4.7.1.2. Stage-1.................................................................................................. 322
4.7.2. Summary ..................................................................................................... 325
4.7.3. Discussion ................................................................................................... 325
4.8. NALP3 candidate region .................................................................................... 331
4.8.1. Association Study ........................................................................................ 331
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4.8.1.1 Tagging SNP selection .......................................................................... 331
4.8.1.1 Stage-1.................................................................................................. 334
4.8.2. Summary ..................................................................................................... 334
4.8.3. Discussion ................................................................................................... 334
4.9. Association analysis of the susceptibility alleles according to response to IL-1
blockade ........................................................................................................................ 340
4.9.1. Discussion ....................................................................................................... 341
5. Discussion ............................................................................................................ 346
6. Future Work ......................................................................................................... 352
Appendix 1. Golden Gate genotyping platform ............................................................ 398
A. Methodology ............................................................................................................ 398
B. Genotyping Protocol ................................................................................................. 400
Appendix 2. Genotyping assay results of Stage-1 SNPs ................................................ 402
Appendix 3. IL-18BP haplotype transient transfection assays ....................................... 410
A. Methods ................................................................................................................... 410
B. Results ...................................................................................................................... 412
Appendix 4. Publications ................................................. Error! Bookmark not defined.
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Table of Tables
Table 1.1 Features of the JIA subtypes according to the ILAR classification ....................... 32
Table 1.2 Differential diagnosis of sJIA .............................................................................. 35
Table 3.1 Flanking sequences scanned for PGA variation discovery .................................... 87
Table 3.2 CEPH samples in the population genotyping databases ........................................ 92
Table 3.3 BSPAR repository patient samples used in GoldenGate Pilot Study ................... 102
Table 4.1 BSPAR repository patient GoldenGate Pilot Study ............................................ 141
Table 4.2 Genotyped SNPs classed as failed assays ........................................................... 142
Table 4.3 Genotyped SNPs significantly deviating from Hardy-Weinberg Equilibrium ..... 147
Table 4.4 Genotyping discrepancies between the two genotyping platforms ...................... 148
Table 4.5 Comparison of LD (r2) values from different population genotyping data sets ... 157
Table 4.6 Comparison of SNP frequencies in different population genotyping data sets .... 160
Table 4.7 IL18BP candidate region tSNPs ......................................................................... 167
Table 4.8 IL18BP candidate region stage-1 analysis results ............................................... 168
Table 4.9 IL18BP candidate region stage-2 analysis results ............................................... 171
Table 4.10 IL18BP candidate region pooled cohorts analysis results.................................. 172
Table 4.11 IL18BP candidate region analysis results with WTCCC2 controls.................... 174
Table 4.12 Additional SNPs tagged by IL18BP SNP1 ....................................................... 176
Table 4.13 IL18BP SNP1 and 2 genotypes of WC healthy controls ................................... 188
Table 4.14 IL-18BP protein expression levels from IFNγ stimulated PBMCs .................... 192
Table 4.15 IL18BP RNA expression levels from IFNγ stimulated PBMCs ........................ 199
Table 4.16 IL1RAP candidate region tSNPs ....................................................................... 216
Table 4.17 Significant IL1RAP candidate region stage-1 analysis results ........................... 217
Table 4.18 Non-significant IL1RAP candidate region stage-1 analysis results .................... 220
Table 4.19 Conditional analysis of associated IL1RAP region SNPs within LD clusters .... 230
Table 4.20 Conditional analysis of associated IL1RAP region SNPs between LD clusters . 233
Table 4.21 IL1RAP candidate region stage-2 analysis results ............................................. 234
Table 4.22 IL1RAP candidate region stratified analysis results .......................................... 236
Table 4.23 IL1RAP candidate region analysis results with WTCCC2 controls ................... 238
Table 4.24 Additional SNPs tagged by IL1RAP SNP14 ..................................................... 239
Table 4.25 IL1 ligand cluster candidate region tSNPs ........................................................ 251
Table 4.26 IL1 ligand cluster additional tagging of analysis excluded SNPs ...................... 252
Table 4.27 Significant IL1 ligand cluster candidate region stage-1 analysis results ............ 254
Table 4.28 Non-significant IL1 ligand cluster candidate region stage-1 analysis results ..... 258
Table 4.29 Conditional analysis of associated IL1 ligand region SNPs within LD clusters . 264
Table 4.30 Conditional analysis of associated IL1 ligand region SNPs between LD clusters
......................................................................................................................................... 265
Table 4.31 IL1 ligand candidate region stage-2 analysis results ......................................... 266
Table 4.32 IL1 ligand cluster candidate region stratified analysis results ........................... 267
Table 4.33 IL1 ligand candidate region analysis results with WTCCC2 controls ................ 269
Table 4.34 Conditional analysis of associated IL1 ligand region SNPs with WTCCC2
controls ............................................................................................................................. 271
Table 4.35 Additional SNPs tagged by IL1 ligand cluster SNPs ........................................ 273
Table 4.36 IL1 receptor cluster candidate region tSNPs ..................................................... 287
Table 4.37 IL1 receptor cluster additional tagging of analysis excluded SNPs ................... 288
Table 4.38 Significant IL1 receptor cluster candidate region stage-1 analysis results ......... 289
Table 4.39 Non-significant IL1 receptor cluster candidate region stage-1 analysis results .. 294
Table 4.40 Conditional analysis of associated IL1 receptor region SNPs ........................... 300
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