Genetic determinants of vitamin D status and susceptibility to acute respiratory infection

286 Pages·2017·16.26 MB·English

Genetic determinants of vitamin D status and susceptibility to
acute respiratory infection

David Anthony Jolliffe
Queen Mary, University of London
Submitted in partial fulfilment of the requirements of the degree of Doctor of Philosophy

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Statement of Originality

I, David Anthony Jolliffe, confirm that the research included within this thesis is my own work or that
where it has been carried out in collaboration with, or supported by others, that this is duly
acknowledged below and my contribution indicated. Previously published material is also
acknowledged below.

I attest that I have exercised reasonable care to ensure that the work is original, and does not to the
best of my knowledge break any UK law, infringe any third party’s copyright or other Intellectual
Property Right, or contain any confidential material.

I accept that the College has the right to use plagiarism detection software to check the electronic
version of the thesis.

I confirm that this thesis has not been previously submitted for the award of a degree by this or any
other university.

The copyright of this thesis rests with the author and no quotation from it or information derived from
it may be published without the prior written consent of the author.

Signature:

Date: 29th February 2016

Details of collaborations:

I worked with the three clinical trial study teams from the outset and aided in the recruitment and
follow-up of participants, which included: collection of anthropometric, lifestyle, and disease
phenotype data by questionnaire; collection of disease phenotype data by clinical procedures e.g. lung
function and quadriceps strength tests; collection, separation, and storage of blood samples; and
collection of induced sputum samples.

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I selected my panel of SNP, designed custom TaqMan assays, and conducted the genotyping of clinical
trial participants under the supervision of Dr Mimoza Hoti (Queen Mary University’s Genome Centre).

In collaboration with Dr Jennifer Roe (Division of Infection & Immunity, University College London) I
carried out RNA extraction and RT-qPCR on a subset of participants from the ViDiCO trial.

I assisted Dr. Claire Greiller (Blizard Institute, QMUL, UK) in the ex vivo stimulation of whole blood
samples, and aspiration of supernatants for freezer storage and subsequent 30-plex ELISAs. I also
conducted whole blood and PBMC assays on clinical trial samples collected in Dr. Greiller’s absence.

Biochemical analyses for the 3 clinical trials to determine serum concentrations of 25-hydroxyvitamin
D, albumins, calcium, and parathyroid hormone were carried out in the Department of Clinical
Biochemistry at Homerton Hospital, by Ms Marion Rowe and Dr Peter Timms.

Primary analysis of clinical trial results (time to ARI/exacerbation) was performed by Professor Adrian
Martineau (Blizard Institute, QMUL, UK)

All cross-sectional, main effects, interaction and meta-analyses presented in this thesis were
performed by myself.

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Abstract.

Introduction: Acute respiratory infections (ARI) are a major global cause of morbidity and mortality.
Vitamin D deficiency has been reported to associate with susceptibility to ARI and with greater severity
and poorer control of asthma and chronic obstructive pulmonary disease (COPD). Clinical trials of
vitamin D for the prevention of ARI have yielded heterogeneous results, with some showing protection
and others not. This may reflect variation in the frequency of genetic variants influencing response to
vitamin D supplementation in different populations. The impact that genetic variation in the vitamin
D pathway has on vitamin D status, disease phenotype and response to vitamin D supplementation in
prevention of ARI has not been comprehensively investigated.
Methods: I conducted:
1. A systematic review and meta-analysis of clinical studies which have investigated vitamin D as a
potential therapy for ARI;
2. Three cross-sectional studies (in n=297 adult asthma patients, n=278 COPD patients, and n=272
older adults) to investigate potential environmental determinants (lifestyle and anthropometric) and
genetic determinants (35 single nucleotide polymorphisms [SNP] in 11 vitamin D related genes) of
serum 25-hydroxyvitamin D concentration (25[OH]D) and clinical phenotype;
3. Three prospective studies investigating the influence of genetic variation in the vitamin D pathway
on a) susceptibility to ARI (main effects analysis) and b) efficacy of vitamin D supplementation for the
prevention of ARI (interaction analysis).
Results: My systematic review identified consistent reports of an inverse association between vitamin
D status and risk of ARI in observational studies, and heterogeneous reports from clinical trials. My
cross-sectional studies identified a range of classical environmental factors which predict vitamin D
status in the three study populations, but did not identify any genetic variants in the vitamin D
pathway that associate with vitamin D status. I identified an association between vitamin D deficiency
and decreased lung function in COPD patients, but no associations between vitamin D deficiency and
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asthma phenotype. Finally, my analysis identified a haplotype of 5 single nucleotide polymorphisms
in the vitamin D receptor (VDR) gene which significantly modify the effect of vitamin D
supplementation on risk of upper respiratory infection in COPD patients.
Conclusions: I identified environmental determinants that predict 25(OH)D concentrations in all three
study populations, but only found an association between vitamin D deficiency and disease severity
in COPD patients. Furthermore, I identified a haplotype in VDR which modifies the effect of vitamin D
supplementation in COPD patients to result in a significantly reduced risk of ARI.

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Table of Contents.

Abstract. ............................................................................................................................................................4
List of abbreviations. .......................................................................................................................................14
1. Introduction. ..................................................................................................................................17
1.1. Acute Respiratory Infection............................................................................................................18
1.1.1. Definition, Classification and Aetiology of ARI. .............................................................................18
Upper respiratory infection. ...........................................................................................................18
Lower respiratory infection. ...........................................................................................................19
1.1.2. Pathology of ARI .............................................................................................................................20
1.1.3. The Burden of ARI: Economics & Mortality. ..................................................................................21
1.2. Vitamin D. .......................................................................................................................................25
1.2.1. Biochemistry and metabolism. ......................................................................................................25
1.2.2. Vitamin D deficiency & susceptibility to Acute Respiratory Infections..........................................28
1.2.3. Immunomodulatory actions of vitamin D. .....................................................................................29
Effect of vitamin D on innate immune responses. .........................................................................29
Effect of vitamin D on adaptive immune responses. .....................................................................32
1.2.4. Immune dysregulation in older adults. ..........................................................................................32
1.2.5. Immune dysregulation in Asthma & COPD patients......................................................................33
1.2.6. Clinical studies of vitamin D and ARI. ............................................................................................35
1.3. Vitamin D Pathway Genes. ............................................................................................................36
1.3.1. Metabolism. ...................................................................................................................................37
1.3.2. Transport. .......................................................................................................................................41
1.3.3. Signalling. .......................................................................................................................................43
1.4. Summary. .......................................................................................................................................47
1.5. Hypothesis and Objectives of the project. .....................................................................................48
2. Methods. ........................................................................................................................................50
2.1. Systematic review of vitamin D and ARI studies............................................................................50
2.1.1. Search method. ..............................................................................................................................50
2.1.2. Exclusion/Inclusion Criteria. ...........................................................................................................50
2.2. Clinical trials. ..................................................................................................................................51
2.2.1. Participants. ...................................................................................................................................51
2.2.2. Screening Procedures. ....................................................................................................................57
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2.2.3. Definition of respiratory outcomes. ...............................................................................................58
2.2.4. Investigational medicinal product (IMP). ......................................................................................60
2.3. Laboratory analyses. ......................................................................................................................61
2.3.1. Biochemistry analysis. ....................................................................................................................61
2.3.2. Genotyping. ....................................................................................................................................61
2.3.3. Immunological analysis. .................................................................................................................68
2.3.4. Gene expression analysis. ..............................................................................................................69
2.4. Statistical analyses. ........................................................................................................................74
2.4.1. Cross-sectional analyses. ...............................................................................................................74
2.4.2. Main effects analyses. ....................................................................................................................75
2.4.3. Interaction analyses. ......................................................................................................................75
2.4.4. Meta-analysis. ................................................................................................................................76
3. Vitamin D in the prevention of acute respiratory infections: Systematic review of clinical
studies. ........................................................................................................................................................78
3.1. Introduction. ...................................................................................................................................79
3.2. Results. ...........................................................................................................................................80
3.2.1. Identification and Selection of Studies...........................................................................................80
3.2.2. Study Characteristics. .....................................................................................................................81
3.2.3. Study Findings. ...............................................................................................................................81
3.2.4. Aggregate data meta-analysis of clinical trials. ............................................................................90
3.3. Discussion. ......................................................................................................................................91
3.3.1. Observational Studies: Strengths and Limitations. .......................................................................91
3.3.2. Intervention Studies: Strengths and Limitations. ..........................................................................92
3.3.3. Meta-analysis of clinical trials. ......................................................................................................94
3.4. Conclusions. ....................................................................................................................................94
4. Determinants and clinical correlates of vitamin D status in adults with asthma. .....................96
4.1. Introduction. ...................................................................................................................................96
4.2. Results. ...........................................................................................................................................98
4.2.1. Study population. ...........................................................................................................................98
4.2.2. Environmental determinants of serum 25(OH)D concentration. ............................................... 100
4.2.3. Genetic determinants of serum 25(OH)D concentration. ........................................................... 102
4.2.4. Association between vitamin D status and asthma phenotype. ................................................ 104
4.2.5. Association between genetic factors and asthma phenotype. .................................................. 111
4.3. Discussion. ................................................................................................................................... 122
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4.3.1. Study Strengths. .......................................................................................................................... 123
4.3.2. Study Limitations. ........................................................................................................................ 124
4.4. Conclusions. ................................................................................................................................. 124
5. Cross-sectional analysis: Prevalence, determinants and clinical correlates of vitamin D
deficiency in Chronic Obstructive Pulmonary Disease. ............................................................................. 136
5.1. Introduction. ................................................................................................................................ 136
5.2. Results. ........................................................................................................................................ 138
5.2.1. Study population. ........................................................................................................................ 138
5.2.2. Environmental determinants of serum 25(OH)D concentration. ............................................... 140
5.2.3. Genetic determinants of serum 25(OH)D concentration. ........................................................... 142
5.2.4. Association between vitamin D status and COPD phenotype. ................................................... 144
5.2.5. Association between genetic factors and COPD phenotype. ..................................................... 153
5.3. Discussion. ................................................................................................................................... 162
5.3.1. Study strengths. .......................................................................................................................... 164
5.3.2. Study limitations. ........................................................................................................................ 165
5.3. Conclusions. ................................................................................................................................. 165
6. Cross-sectional analysis: Environmental and genetic determinants of vitamin D status among
older adults in London, UK. ........................................................................................................................ 125
6.3. Introduction. ................................................................................................................................ 125
6.4. Results. ........................................................................................................................................ 127
6.4.1. Study population. ........................................................................................................................ 127
6.4.2. Environmental determinants of vitamin D status. ..................................................................... 129
6.5. Discussion. ................................................................................................................................... 133
6.5.1. Study strengths. .......................................................................................................................... 134
6.5.2. Study limitations. ........................................................................................................................ 134
6.6. Conclusions. ................................................................................................................................. 135
7. Genetic variants in the vitamin D pathway and susceptibility to acute respiratory infections in
patients with asthma. ................................................................................................................................. 167
7.1. Introduction. ................................................................................................................................ 167
7.2. Results. ........................................................................................................................................ 170
7.2.1. Main effects: does vitamin D pathway genotype influence risk of URI or asthma exacerbation,
independent of vitamin D supplementation? .............................................................................................. 170
7.2.2. Interaction analysis: does vitamin D pathway genotype influence effect of vitamin D
supplementation in prevention of URI or asthma exacerbation? ............................................................... 175
7.3. Discussion. ................................................................................................................................... 182
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7.3.1. Study strengths. .......................................................................................................................... 183
7.3.2. Study limitations. ........................................................................................................................ 184
7.4. Conclusions. ................................................................................................................................. 184
8. Genetic variants in the vitamin D pathway and susceptibility to acute respiratory infections in
older adults. ................................................................................................................................................. 185
8.1. Introduction. ................................................................................................................................ 185
8.2. Results. ........................................................................................................................................ 187
8.2.1. Main effects analysis: does vitamin D pathway genotype influence risk of ARI, independent of
vitamin D supplementation? ........................................................................................................................ 187
8.2.2. Interaction analysis: does genotype influence effect of vitamin D supplementation in prevention
of ARI? ..................................................................................................................................................... 192
8.3. Discussion. ................................................................................................................................... 197
8.3.1. Study strengths. .......................................................................................................................... 198
8.3.2. Study limitations. ........................................................................................................................ 198
8.4. Conclusions. ................................................................................................................................. 199
9. Genetic variants in the vitamin D pathway and susceptibility to acute respiratory infections in
patients with COPD. .................................................................................................................................... 200
9.1. Introduction. ................................................................................................................................ 200
9.2. Results. ........................................................................................................................................ 202
9.2.1. Main effects: does vitamin D pathway genotype influence risk of URI or COPD exacerbation,
independent of vitamin D supplementation? .............................................................................................. 202
9.2.2. Interaction analysis: does genotype influence effect of vitamin D supplementation in prevention
of URI or COPD exacerbation? ..................................................................................................................... 207
9.2.3. SNP haplotype analysis. .............................................................................................................. 215
9.2.4. Immune profile analysis. ............................................................................................................. 220
9.2.5. VDR expression analysis. ............................................................................................................. 224
9.3. Discussion. ................................................................................................................................... 225
9.3.1. Study strengths. .......................................................................................................................... 227
9.3.2. Study Limitations. ........................................................................................................................ 227
9.4. Conclusions. ................................................................................................................................. 228
10. Concluding remarks and future directions for research. ......................................................... 229
References. .......................................................................................................................................... 238
Appendix: Publications arising from this thesis. ................................................................................. 258

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List of Tables

Table 2.1: ViDiFlu trial dosing regimens 57
Table 2.2: Single nucleotide polymorphisms (SNPs) identified as putative modifiers of the
effects of vitamin D supplementation 63

Table 2.3. Pearson’s Chi-squared test results for Hardy-Weinberg equilibrium in n=835
genotyped SNP 67

Table 2.4. Preparation of TLR ligands used to stimulate cytokine release in whole blood
samples 68

Table 3.1. Cross-sectional studies investigating association between vitamin D status
and susceptibility to ARI 83

Table 3.2. Case-control studies investigating association between vitamin D status and
susceptibility to ARI 84

Table 3.3. Cohort studies investigating association between vitamin D status and
susceptibility to ARI 85

Table 3.4. Clinical trials investigating effects of vitamin D supplementation on incidence
of ARI 88

Table 4.1: Participant Characteristics (ViDiAs trial) 99

Table 4.2: Environmental determinants of vitamin D status in asthma patients 101

Table 4.3: Genetic determinants of vitamin D status in asthma patients 103

Table 4.4. Determinants of Asthma Control Test (ACT) score 105

Table 4.5. Determinants of % predicted Forced Expiratory Volume in 1 second (FEV ) 106
1

Table 4.6. Determinants of inhaled corticosteroid (ICS) dose 108

Table 4.7. Determinants of fractional exhaled nitric oxide (FeNO) 109

Table 4.8. Determinants of sputum eosinophilia (subset of n=35 participants) 110

Table 5.1 Participant Characteristics (ViDiCO trial) 139

Table 5.2. Environmental determinants of serum 25-hydroxyvitamin D concentration in
COPD patients 141

Table 5.3. Genetic determinants of serum 25-hydroxyvitamin D concentration in COPD
Patients 143

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