Molecular Biomarker Discovery in Psoriatic Arthritis

216 Pages·2016·5.02 MB·English

Molecular Biomarker Discovery in Psoriatic Arthritis
by
Remy Angela Pollock
A thesis submitted in conformity with the requirements
for the degree of Doctor of Philosophy
Institute of Medical Science
University of Toronto
© Copyright by Remy Angela Pollock 2016
Molecular Biomarker Discovery in Psoriatic Arthritis
Remy Angela Pollock
Doctor of Philosophy
Institute of Medical Science
University of Toronto
2016
Abstract
Aim: Psoriatic arthritis (PsA) is an inflammatory arthritis of unknown etiology that develops in
approximately 30% of individuals with psoriasis. No objectively measurable biomarker has been
identified for PsA, due in part to insufficient knowledge of its etiopathogenesis. This work aims
to identify candidate biomarkers of PsA by studying its underlying transcriptomic and
epigenomic mechanisms.
Methods: Psoriasis (PsC) and PsA patients from a prospective cohort were analyzed. Whole
blood, serum, and semen samples were obtained from subsets of patients and unaffected controls
for transcriptomic, protein, and epigenomic analyses, respectively. Potential epigenetic
mechanisms were also analyzed using self-reported family history data from the entire PsC and
PsA cohort to further explore the parent-of-origin effect.
Results: Transcriptomic analyses identified several genes involved in innate immunity,
particularly toll-like receptor signalling as differentially expressed in whole blood of PsA and
PsC patients. Four candidate gene expression biomarkers CXCL10, NOTCH2NL, HAT1, and
SETD2 were replicated in an independent cohort of PsC and PsA patients. Soluble CXCL10 was
significantly elevated in baseline serum samples of psoriasis patients who later developed PsA
compared to patients who did not develop PsA. Excessive paternal transmission was found in
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PsC and PsA patients, as well as genetic anticipation manifesting as increased disease severity
during male transmission. DNA methylation profiling of sperm cells revealed several germ line
variations associated with psoriasis and PsA occurring near or within genes involved in
inflammatory and immune system processes, including HCG26 within the major
histocompatibility complex.
Conclusions: Whole blood transcriptomic and serum protein analysis identified the chemokine
CXCL10 as a putative predictive biomarker of PsA in PsC patients. Evidence of a parent-of-
origin effect, genetic anticipation, and the identification of germ line DNA methylation
variations in patients suggest a role for epigenetic mechanisms in psoriatic disease
etiopathogenesis, and a potential new avenue of biomarker discovery.
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Acknowledgments

First and foremost, I would like to express my gratitude to my supervisor and mentor Dr. Dafna
Gladman for inspiring me to pursue research in this field and the endless guidance and support
she provided over the past several years. To my committee members, Drs. Cathy Barr, Jo Knight,
and Art Petronis, thank you for providing direction, insight, knowledge, and encouragement
during the completion of my degree. I would also like to thank my predecessors Drs. Vinod
Chandran and Lihi Eder, for setting high standards of scholarship that I continuously strive to
emulate; colleagues Fawnda Pellett and Fatima Abji for their technical expertise and support in
designing and performing laboratory-based analyses; collaborators Drs. Proton Rahman and Kun
Liang for providing data and analytical advice; Anne MacKinnon and the staff of the University
of Toronto Psoriatic Arthritis Program for administrative and clinical support; and finally, the
patients of the University of Toronto Psoriatic Arthritis Program whose contributions made these
studies possible.
I would like to acknowledge the Canadian Institutes of Health Research for funding my work
through the Frederick Banting and Charles Best Canada Graduate Scholarship Doctoral Research
Awards, as well as the Arthritis Research Foundation, National Psoriasis Foundation, and
Krembil Foundation for providing funds for the germ line methylation study.
Lastly, I would like to thank my parents, Hume and Bella, who encouraged me from a young age
to pursue science, and my fiancé Colin, whom I met on the first day I started this degree, and
whose confidence in me and unconditional support from that day forward made all the
difference.

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Contributions

For Chapter 3, I was responsible for processing and extracting the RNA samples, performing
secondary bioinformatics analyses, data mining, and performing the technical validation by
qPCR. I was also responsible for the statistical analysis of the nCounter® data, purification of
leukocyte subsets, RNA extraction from purified cells, and measurement of candidate gene
expression by qPCR. I generated all figures in Chapter 3 with the exception of Figures 3.1 and
3.2, which were created by Kun Liang (Department of Statistics and Actuarial Science,
University of Waterloo). For Chapter 4, I contributed to the processing and biobanking of serum
samples from psoriasis patients, performed all of the statistical analyses and interpretation of
CXCL10 protein expression data, and was responsible for designing and acquiring data for the
gene expression experiments. Figures in Chapter 4 were created by Fatima Abji (Psoriatic
Arthritis Program, Toronto Western Research Institute), with the exception of Figure 4.3 which I
created. For Chapter 5, I was responsible for gathering all of the family history data from various
clinical databases, verifying data with the baseline research protocols, family history
questionnaires, patient charts, or by telephone interviews with patients, and performing all data
analysis and interpretation. For Chapter 6, I was responsible for patient recruitment, sample
collection and processing, DNA extraction and preparation of DNA samples for arrays. I also
performed all steps of data quality control, preprocessing, statistical/bioinformatics analyses, and
created all figures in this chapter.

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Table of Contents
Acknowledgments .......................................................................................................................... iv
Contributions ................................................................................................................................... v
Table of Contents ........................................................................................................................... vi
Abbreviations ............................................................................................................................... viii
List of Tables .................................................................................................................................. x
List of Figures ............................................................................................................................... xii
List of Appendices ....................................................................................................................... xiv
Chapter 1 Literature Review ........................................................................................................... 1
1.1 Psoriasis .............................................................................................................................. 1
1.2 Psoriatic Arthritis ................................................................................................................ 7
1.3 Tools for Diagnosing PsA ................................................................................................. 14
1.4 Molecular Biomarkers of PsA .......................................................................................... 23
Rationale, Hypotheses and Specific Aims ................................................................... 47
2.1 Rationale ........................................................................................................................... 47
2.2 Hypotheses and Specific Aims ......................................................................................... 48
............................................................................................................. 50
3.1 Introduction ....................................................................................................................... 50
3.2 Materials and Methods ...................................................................................................... 51
3.3 Results ............................................................................................................................... 55
3.4 Discussion ......................................................................................................................... 82
.................................................................... 86
4.1 Introduction ....................................................................................................................... 86
4.2 Materials and Methods ...................................................................................................... 87
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4.3 Results ............................................................................................................................... 90
4.4 Discussion ......................................................................................................................... 99
Further Evidence Supporting a Parent-of-Origin Effect in Psoriatic Disease ............ 103
5.1 Introduction ..................................................................................................................... 103
5.2 Patients and Methods ...................................................................................................... 104
5.3 Results ............................................................................................................................. 105
5.4 Discussion ....................................................................................................................... 112
.................................... 115
6.1 Introduction ..................................................................................................................... 115
6.2 Methods ........................................................................................................................... 117
6.3 Results ............................................................................................................................. 121
6.4 Discussion ....................................................................................................................... 141
General Discussion ..................................................................................................... 146
7.1 Limitations ...................................................................................................................... 158
7.2 Conclusions ..................................................................................................................... 163
7.3 Future Directions ............................................................................................................ 164
Appendix ..................................................................................................................................... 167
References ................................................................................................................................... 178

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Abbreviations
APCA Anti-citrullinated peptide antibody
AS Ankylosing spondylitis
AUC Area under the curve
Avy Agouti viable yellow
AxinFU Axin fused
BMI Body mass index
CASPAR Classification of Psoriatic Arthritis
CD Cluster of differentiation
CI Confidence interval
CpG Cytosine-guanine dinucleotide
CRP C-reactive protein
CXCL10 C-X-C motif ligand 10
DEG Differentially expressed gene
DMARD Disease-modifying anti-rheumatic drug
DMR Differentially methylated region
DNMT DNA methyltransferase
DZ Dizygotic
ELISA Enzyme-linked immunosorbent assay
ESR Erythrocyte sedimentation rate
FC Fold change
FDR False discovery rate
GAPDH Glyceraldehyde 3-phosphate dehydrogenase
GWAS Genome-wide association study
H3K4/9/27/36 Histone 3 lysine 4/9/27/36
HAT1 Histone acetyltransferase 1
HCG26 HLA complex group 26
HLA Human leukocyte antigen
HNPCC Hereditary non-polyposis colorectal cancer
IAP Intracisternal A particle retrotransposon
ICR Imprinting control region
IFN Interferon
Ig Immunoglobulin
IL Interleukin
IQR Interquartile range
KIR Killer cell immunoglobulin-like receptor
MAF Minor allele frequency
M-CSF Monocyte colony stimulating factor
mDC Myeloid dendritic cell
MHC Major histocompatibility complex
MICA/B MHC Class I polypeptide-related sequence A/B
MMP Matrix metalloproteinase
mRNA Messenger RNA
MS Multiple sclerosis
MTX Methotrexate
MZ Monozygotic
viii
ncRNA Non-coding RNA
NF-κB Nuclear factor kappa B
NK Natural killer cell
NOTCH2NL Notch 2 N-terminal like
NSAID Non-steroidal anti-inflammatory drug
OCP Osteoclast precursor
OR Odds ratio
PASE Psoriatic Arthritis Screening and Evaluation Tool
PASI Psoriasis area and severity index
PAQ Psoriasis Assessment Questionnaire
PBMC Peripheral blood mononuclear cell
pDC Plasmacystoid dendritic cell
PEST Psoriasis Epidemiology Screening Tool
PE Phycoerythrin
PsA Psoriatic arthritis
PsC Cutaneous psoriasis without arthritis
qPCR Quantitative PCR
RA Rheumatoid arthritis
RANKL Receptor activator of NF-κB ligand
RF Rheumatoid factor
RNA Ribonucleic acid
ROC Receiver operating characteristics
SD Standard deviation
SETD2 SET domain containing 2
SLE Systemic lupus erythematosus
SNP Single nucleotide polymorphism
T1D Type 1 diabetes
Th1/2/17 T helper type 1/2/17
TLR Toll-like receptor
TNFa Tumour necrosis factor alpha
ToPAS Toronto Psoriatic Arthritis Screen
TP, TN, FP, FN True positive, true negative, false positive, false negative

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List of Tables
Table 1.1. Performance characteristics of diagnostic tools.
Table 3.1. Demographic and clinical characteristics of the discovery and replication cohorts.
Table 3.2. Enriched biological annotations among the 494 differentially expressed genes between
PsA and PsC.
Table 3.3. Top differentially expressed genes between PsA and PsC from primary microarray
analyses.
Table 3.4. Differentially expressed genes between PsA compared to PsC identified by TLR
signaling and chromatin modification targeted qPCR arrays.
Table 3.5. Candidate genes selected for replication testing in an independent cohort by
nCounter® technology.
Table 3.6. Correlations between gene expression and clinical variables from Table 3.1 that differ
between discovery and replication cohorts.
Table 3.7. Comparison of clustered and unclustered PsA patients in the validation cohort.
Table 4.1. Demographic and clinical characteristics of the study subjects at baseline.
Table 4.2. Baseline CXCL10 as a predictor of PsA converter status.
Table 4.3. Baseline CXCL10 compared to clinical predictors of conversion of PsA.
Table 5.1. Cross tabulation of disease status in fathers and mothers of all probands.
Table 5.2. Cross tabulation of disease status in fathers and mothers of the PsA probands.
Table 5.3. Cross tabulation of disease status in fathers and mothers of the PsC probands.
Table 5.4. Results of univariate logistic regression models examining the association between
paternally-transmitted disease and clinical and genetic variables in PsA patients from
Newfoundland.
Table 5.5. Significant results from multivariable logistic regression models examining the
association between paternally-transmitted disease and clinical and genetic variables, adjusted
for sex of the proband.
Table 6.1 Demographic and clinical characteristics of the study subjects.
Table 6.2 Biological functional enrichment analysis of all genes found to be differentially
methylated sperm cells.
Table 6.3 Top hyper and hypomethylated genes from each of the groupwise comparisons and
genes most relevant to psoriatic disease.
x