The Immunogenetics of Asthma Exacerbations in Children · The Immunogenetics of Asthma...

The Immunogenetics of Asthma Exacerbations

in Children

Joelene Ann Bizzintino B.Sc. (Hons)

This thesis is presented for the degree of

Doctor of Philosophy

at The University of Western Australia,

School of Paediatrics and Child Health.

2011

i

”Apply your heart to instruction

and your ears to words of knowledge”

Proverbs 23:12

ii

DEDICATION

This work is dedicated to

Theodore Box

a truly great man who has always inspired me and imparted countless words of wisdom.

He has taught me many things that I will never forget,

the most important of which is

“always think for yourself”

and whilst I have drawn from the support and expertise of supervisors and colleagues,

this thesis is definitely written proof of that.

iii

DECLARATION FOR THESES CONTAINING PUBLISHED WORK AND/OR

WORK PREPARED FOR PUBLICATION

This thesis contains published work and/or work prepared for publication, some of

which has been co-authored. The bibliographical details of the work and where it

appears in the thesis are outlined below.

1) Bizzintino J and Lee W-M, Laing IA, Vang F, Pappas T, Zhang G, Martin AC,

Khoo S-K, Cox DW, Geelhoed GC, McMinn PC, Goldblatt J, Gern JE, Le Souëf

PN

Association between human rhinovirus C and severity of acute asthma in

children. Published in: European Respiratory Journal. 2011;37:1037-42.

This constitutes Chapter 2 of this thesis. J Bizzintino was involved in the

recruitment of patients, sample collection, data collection, detection, and typing

assays and was responsible for data analysis and drafting the article. W-M Lee

developed and was responsible for the molecular detection and typing assays,

which was assisted by F Vang, T Pappas and S-K Khoo. IA Laing was involved

in the study design, initiation and management of the study and revision of the

manuscript. G Zhang supported the data analysis. AC Martin was primarily

responsible for the recruitment of patients, sample and data collection, and was

assisted by DW Cox. GC Geelhoed and PC McMinn contributed to study

design, as did JE Gern who also manages the team that performed the detection

and typing assays on the nasal aspirates. J Goldblatt and PN LeSouëf initiated

the study, contributed to its design, data analysis and drafting of the article.

iv

2) JA Bizzintino, C McLean, G Zhang, S-K Khoo, L Subrata, AC Martin, K

Rueter, CM Hayden, A Sharafi, GC Geelhoed, J Goldblatt, PG Holt, IA Laing

and PN LeSouëf

Polymorphisms in Toll-Like Receptor (TLR) genes influence susceptibility

and severity of acute asthma in children

This constitutes Chapter 3 of this thesis and the manuscript has been prepared

for publication. J Bizzintino was involved in study design, the recruitment of

patients, sample collection, data collection, DNA extraction, genotyping and was

responsible for data analysis and writing the article. C McLean was partially

responsible for genotyping and contributed to the study design. G Zhang

assisted the statistical analysis, as did IA Laing who was involved in

experimental design and manuscript revision. S-K Khoo assisted the genotyping

and data collection. L Subrata conducted micro-array and qRT-PCR analyses.

AC Martin, K Rueter and A Sharafi also conducted patient recruitment and

assessments. GC Geelhoed, J Goldblatt, PG Holt, CM Hayden and PN LeSouëf

contributed to the study design.

3) JA Bizzintino, G Zhang, C McLean, S-K Khoo, L Subrata, AC Martin, K

Rueter, CM Hayden, A Sharafi, GC Geelhoed, J Goldblatt, PG Holt, PN

LeSouëf and IA Laing

Polymorphisms in RIG-I-like Receptor Pathway Genes Involved in the

Innate Immune Response to Rhinovirus Affect Susceptibility and Severity

of Acute Asthma in Children.

This constitutes chapter 4 of this thesis and manuscript has been prepared for

publication. J Bizzintino was primarily responsible for study design, performed

all statistical analyses, wrote the manuscript, was involved in patient

recruitment, and performed DNA extraction and genotyping. G Zhang supported

the statistical analysis. C McLean and S-K Khoo performed some of the

genotyping. L Subrata was responsible for micro-array and qRT-PCR analyses.

AC Martin, K Rueter and A Sharafi were all involved with patient recruitment

and assessments. GC Geelhoed, J Goldblatt, PG Holt and PN LeSouëf initiated

the study, and assisted revision of the article. IA Laing supported data analysis,

was involved in initiation of the study, experimental design, and manuscript

revision.

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4) JA Bizzintino, LS Subrata, G Zhang, S-K Khoo, J Goldblatt, PG Holt, GC

Geelhoed, PN LeSouëf and IA Laing.

Genotype-driven expression of novel genes involved in childhood acute

asthma, identified by micro-array.

This constitutes Chapter 5 of this thesis and manuscript has been prepared for

publication. J Bizzintino was responsible for data analysis and writing the

article, was involved in study design, the recruitment of patients, sample

collection, data collection, DNA extraction and genotyping. L Subrata

performed micro-array and qRT-PCR analyses. G Zhang supported statistical

analysis. S-K Khoo assisted genotyping and data collection. J Goldblatt, PG

Holt, GC Geelhoed and PN LeSouëf contributed to study initiation, design and

manuscript revision. IA Laing was involved in study initiation, experimental

design, manuscript revision and supported statistical analysis.

5) JA Bizzintino, LS Subrata, G Zhang, S-K Khoo, J Goldblatt, PG Holt, PN

LeSouëf and IA Laing

Genotypes within steroid responsive genes identified by micro-array are

associated with childhood acute asthma susceptibility, severity and response

to treatment.

This constitutes chapter 6 of this thesis and manuscript has been prepared for

publication. J Bizzintino performed all statistical analyses, wrote the article, was

involved in the recruitment of patients, sample and, data collection, DNA

extraction and genotyping. L Subrata performed micro-array and qRT-PCR

analyses. G Zhang supported the data analysis. S-K Khoo supported genotyping

and data collection. J Goldblatt, PG Holt and PN LeSouëf contributed to the

study design and revision of the paper. IA Laing supported data analysis and

manuscript revision, was involved in initiation of the study and was primarily

responsible for experimental design.

Each author has given permission for the work arising in the above

manuscripts to be presented in this thesis.

______________________ ____________________________

Mrs Joelene Bizzintino Professor Peter Le Souef

PhD Candidate Principal Supervisor

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STATEMENT OF CANDIDATE CONTRIBUTION

The work presented in this thesis was performed at the Institute for Child Health

Research and the School of Paediatrics and Child Health, University of Western

Australia, Princess Margaret Hospital, Subiaco, Perth, Western Australia, under the

supervision of Professor Peter LeSouef, Dr Ingrid Laing and Professor Jack Goldblatt.

With the exception of acknowledged contributions by others, the author performed all

experimental and statistical work presented herein. This thesis is my own account of my

research and has not previously been submitted for a degree at this or any other

University.

Joelene Bizzintino

February 2011

vii

ACKNOWLEDGMENTS

I would like to firstly thank my supervisors, Prof. Peter LeSouef, Dr Ingrid Laing and

Prof. Jack Goldblatt. Peter, your constant support and encouragement, challenging

discussions, fantastic view of the bigger picture and passion for respiratory research

have made you an absolute joy to work with. I have shared some great times overseas

whilst attending international conferences and I thankyou for your willingness to

introduce me to numerous collaborators and help out last-minute when posters don’t

make the trip. I have also immensely appreciated your contagious enthusiasm for

adventure and your great sense of humour that has me laughing out loud (especially in

emails during the early hours of the morning). Ingrid, your understanding and ability to

interpret scientific data will always be a true inspiration for me and I am extremely

fortunate to have learnt a great deal from you during my candidature. I am also grateful

for your willingness to meet up with me to discuss my project, provide feedback on my

work, and set-up meetings or discussions for me with contacts you have made. Jack,

your ability to provide great feedback in a very short time frame or dissolve arguments

with your words of knowledge has always been very impressive and much appreciated.

I’d like to thank the people from our asthma genetics team that I have loved working

with over the years: Kim for your tremendous support, team-player spirit, amazing

generosity, your problem-solving abilities and your true friendship - you have been

absolutely amazing to work with; En nee for your honesty and your faith, Holly for your

constant support and shared love of Edward; Pierre for your humour and valued

friendship; Guicheng for your great statistical support; Catherine for all your good

advice; Carryn for your joy; Des for your passion and humility, Ash for your

enthusiasm; Selma for your kindness and helpfulness; May for your wit, Andrew Martin

for your encouragement and care-free nature; Gareth for being lots of fun to be around;

Jasminka for sharing my stress; Shah for your support that I miss; and Lauren, Olga,

Alicia, Philippa, Michelle and Giovanni for your friendship.

A huge thank you to all those at/affiliated with the School of Paediatrics and Child

Health who have supported and encouraged me and shared some great times during my

candidature. These people include Ruth, Angela, Karla, Clara, Julie, Jen, Sam,

Sunalene, Anthony, Tony, Andrew Currie, Eva, Lea-Ann, Lisa, Gina, Dianne, Steve

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Stick, Erika, Peter Franklin, Michael, Mel, Salina, Catherine Gangell, Jan, Karen and

Bill. In particular, I wish to truly thank Tony for actively emphasising the word

“support” in IT support. And I especially thank my shared-office champs (adopted

sisters, also my adopted brother Patrick) for great friendship and for showing me some

of the best times in my life. Ange, OMG, your help formatting the thesis has been

“gigantic”, and Julie, thanks for your help and attention to detail. Ruth, there aren’t

words. How do you thank someone who goes above and beyond when you need it

most, who constantly encourages you to “just keep swimming” when you are crazy-

overtired or frustrated, and without whom you would not meet an impossible deadline?

I wish to thank my colleagues over the years at the Telethon Institute for Child Health

Research, especially Prof. Wayne Thomas, Belinda, Michael, Jamie, Claire, Lee,

Jacquie, Tracey, Serena, Tatjana, Clint, Paula, Wendy, and Jua, for their individual

contributions and helpful advice.

Many thanks to Dr Wai-Ming Lee, Prof Jim Gern, Tressa, Fue and the team from the

University of Wisconsin-Madison, School of Medicine and Public Health for their

advice and technical assistance regarding the RMA and typing assay. Thank you also

for your friendship, for sharing your laboratory with me and for teaching me new

molecular techniques.

Thankyou Katie Lindsay and Tony Keil for your assistance retrieving samples and

specimen-related data, your constant support is much appreciated.

To my family (Mum, Dad, Poppa, Carrie, Trav, Greg, Kel, Rizzi) and friends (Sonya,

Joe, Dylan, Kelsey, Elina, Fiona, Candace) who loved me, called to encourage me,

prayed for me, helped me, cooked for me, and were very patient with me during my

writing-up stage.

Saving the best for last, I want to thank my awesome husband Carmelo for being very

supportive and loving, for infinite understanding, for his willingness to help wherever

he can and for the best hugs ever. I thank God for you.

ix

ABSTRACT

Acute asthma is an inflammatory disease of the airways that is characterised by a

significant deterioration in respiratory condition and is a primary cause of

hospitalisation for young children. The majority of asthma exacerbations have been

associated with human rhinovirus (HRV) infection. A new and potentially more

pathogenic group of HRVs, called HRVC, has recently been discovered, yet their role in

asthma exacerbations has not been elucidated. In addition, viral recognition pathways

that are crucial for initiating host innate and adaptive immune responses to these viruses

have not been explored for genetic variations that may predispose to acute asthma or

modify disease. The aim of this project was to determine the strains of HRV infecting

children during an asthma attack and to investigate their influence on severity and other

acute asthma phenotypes. In addition, we aimed to study single nucleotide

polymorphisms (SNPs) in genes involved in antiviral defence as well as candidate genes

identified by micro-array of paired acute and convalescent samples for their potential

contribution to asthma exacerbations. We hypothesised that HRVC would be present in

children with acute asthma and cause more severe attacks than other viruses detected.

We also hypothesised that genetic variations in immune response genes and novel

candidates would be associated with acute asthma phenotypes, including susceptibility,

gene expression and exacerbation severity.

Children with acute asthma aged 2-16 years (n=232) were recruited upon presentation

to hospital and followed up after at least six weeks. Asthma exacerbation severity was

assessed in each child and respiratory viruses and HRV strains were identified in 128

nasal aspirates. Gene expression levels were measured by qRT-PCR of peripheral

blood mononuclear cell (PBMC) mRNA from a subset of 50 cases with paired acute

and convalescent samples. Acute asthmatic children were genotyped for 143 SNPs in

34 candidate genes and frequencies were compared with those of 120 non-asthmatic

controls from a longitudinal birth cohort by Chi-squared tests. Genotype/phenotype

associations were assessed by linear or logistic regression adjusted for the potential

effects of gender, age, and/or time lapse between oral steroid administration and blood

collection.

We found that HRV was detected in 87.5% of children, which was higher than

previously reported in children presenting to hospital with acute asthma. HRV typing

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revealed that the recently-discovered HRVC strains were associated with the majority of

these asthma attacks (59.5%) and more severe exacerbations than previously-known

HRV serotypes and other common respiratory viruses. The detection of other

respiratory viruses in children with acute asthma (14.8%) was substantially lower than

the detection rate of HRV and often occurred as HRV co-infections (10.2%). The high

detection rate of HRV precluded comparisons of acute asthma phenotypes between

infected and non-infected children.

Our study of genes involved in the innate immune response to respiratory viruses

including components of both the toll-like receptor (TLR) and retinoic acid inducible

gene (RIG-I)-like receptor (RLR) pathways, identified genetic variants that were related

to acute asthma. Specifically, TLR3, TLR8, DDX58, IFIH1, MAVS, and IFNGR1 genes

contained SNPs that were associated with susceptibility to acute asthma. TLR8, IFIH1,

and MAVS SNPs were associated with mRNA expression levels of encoded products or

downstream pathway proteins. SNPs in TLR7, TLR8, DDX58, and IFNGR1 genes were

associated with exacerbation severity. In particular, for the X chromosome TLR8

rs4830805 G/A SNP, children with homozygous A (girls) or hemizygous A (boys)

genotypes were more common among acute asthmatics than non-asthmatics, had

reduced levels of gene expression during the asthma attack, in convalescence, as well as

differential (acute-convalescent), and suffered more severe attacks than children with

homozygous or hemizygous G genotypes. The polymorphisms investigated in

TICAM1, IL29 and IFNG were not significantly associated with study outcomes in our

children with acute asthma.

For SNPs in novel and steroid-responsive candidate genes that were differentially

expressed according to micro-array analysis of paired acute and convalescent PBMC

mRNA from children with acute asthma, we observed a number of significant

genotype/phenotype relationships. Specific SNPs in MX1, THBS1, STAT4, NLRC4,

TNFSF4 and MNDA were associated with acute asthma susceptibility. SNPs in PF4V1,

CXCL5, KIR2DL4, CXCL1, TNFSF4, AREG, MNDA, NLRC4 and FLT3 were

associated with encoded gene expression levels. Polymorphisms in SPARC, TNFAIP6,

FCER1A, IL23A, IDO1, FPR2, MNDA, NLRC4, TNFSF4 and MS4A4A were associated

with the severity of asthma exacerbations. The STAT4 rs1517352 SNP was associated

with all three outcome measures. Additionally, among the steroid-responsive genes,

SNPs in ALOX15B, FPR2, FLT3, MNDA, AREG and MS4A4A, were associated with a

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child’s response to hospital-administered acute asthma therapy. The MNDA gene

showed associations with all four outcomes relating to acute asthma. For SNPs studied

in LTB4R, THBD, VSIG4 and ADORA3, we did not find any significant relationships

with phenotypes of acute asthma.

In conclusion, our findings suggest that HRVC is by far the most important viral group

detected in children with acute asthma and that the contribution of HRV to exacerbation

severity has been previously underestimated. HRVC may be associated with the

majority of asthma attacks and more severe exacerbations and is therefore of great

clinical relevance and prognostic significance to children with asthma. For the first time

in acute asthma, we have identified genetic variations within antiviral immune and

differentially expressed candidate genes that are likely to play a significant role in

determining a child’s susceptibility to attacks, level of immune response (through gene

expression), exacerbation severity and response to treatment. Altogether, these findings

have the potential to elucidate the mechanisms of childhood acute asthma and facilitate

the development of new preventative and therapeutic strategies.

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TABLE OF CONTENTS

DEDICATION....................................................................................................... ii

DECLARATION FOR THESES CONTAINING PUBLISHED WORK

AND/OR WORK PREPARED FOR PUBLICATION......................................... iii

STATEMENT OF CANDIDATE CONTRIBUTION.......................................... vi

ACKNOWLEDGMENTS................................................................................... vii

ABSTRACT........................................................................................................... ix

TABLE OF CONTENTS....................................................................................... xii

ABBREVIATIONS............................................................................................... xvi

LIST OF FIGURES............................................................................................... xxii

LIST OF TABLES................................................................................................. xxvi

PEER REVIEWED ARTICLES AND PRESENTATIONS…………………….. xxvii

CHAPTER 1: LITERATURE REVIEW............................................................ 1

1.1 INTRODUCTION...................................................................................... 2

1.2 ACUTE ASTHMA...................................................................................... 2

1.3 ACUTE ASTHMA SYMPTOMS............................................................. 3

1.4 DIAGNOSIS OF ACUTE ASTHMA........................................................ 4

1.5 ACUTE ASTHMA SEVERITY................................................................ 5

1.6 PATHOPHYSIOLOGY............................................................................. 8

1.6.1 Airway Obstruction......................................................................... 8

1.6.1.1 Airway inflammation and mucus hypersecretion........... 12

1.6.1.2 Bronchoconstriction....................................................... 13

1.7 ACUTE ASTHMA TREATMENT........................................................... 14

1.7.1 B2 adrenergic receptor (B2AR) agonists........................................ 15

1.7.2 Corticosteroids................................................................................ 16

1.7.3 Anticholinergics.............................................................................. 16

1.7.4 Oxygen therapy............................................................................... 17

1.8 ENVIRONMENTAL AETIOLOGY........................................................ 17

1.8.1 Allergens/Atopy.............................................................................. 18

1.8.2 Irritants, Temperature, Stress and Exercise.................................... 19

1.8.3 Bacterial Respiratory Infection....................................................... 19

1.8.4 Viral Respiratory Infection............................................................. 20

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1.9 HUMAN RHINOVIRUS (HRV) AND ACUTE ASTHMA................... 20

1.9.1 Viral Factors.................................................................................... 21

1.9.2 HRV Groups and Strains................................................................ 22

1.9.3 HRV Detection, Quantitation and Typing...................................... 23

1.9.4 Prevalence, Transmission and Viral Replication............................ 24

1.10 HOST RECOGNITION OF HRV............................................................ 25

1.10.1 Toll-like receptors (TLR)................................................................ 25

1.10.2 Retinoic acid inducible gene I (RIG-I)-like receptors (RLR)......... 29

1.11 HOST RESPONSE TO HRV.................................................................... 31

1.12 ASTHMA GENETICS............................................................................... 34

1.12.1 Methods to identify candidate genes............................................... 34

1.12.1.1 Criteria for candidate gene and polymorphism

selection....................................................................... 34

1.12.2 Identified asthma genes................................................................... 35

1.12.3 Potential candidate genes................................................................ 35

1.12.3.1 Innate Immune Genes Involved in Response to HRV 35

1.12.3.1.1 Toll-like receptors........................................ 35

1.12.3.1.2 RLR Pathway Genes..................................... 40

1.12.3.2 Micro-array identified acute asthma candidate genes. 42

1.12.3.2.1 Novel candidate genes.................................. 43

1.12.3.2.2 Steroid-responsive candidate genes.............. 53

1.13 RESEARCH PROPOSAL......................................................................... 60

1.13.1 Hypotheses...................................................................................... 60

1.13.2 Project Aims.................................................................................... 60

CHAPTER 2: ASSOCIATION BETWEEN HUMAN RHINOVIRUS C

AND SEVERITY OF ACUTE ASTHMA IN CHILDREN... 61

2.1 ABSTRACT................................................................................................ 62

2.2 INTRODUCTION...................................................................................... 63

2.3 MATERIALS AND METHODS............................................................... 64

2.4 RESULTS.................................................................................................... 66

2.5 DISCUSSION.............................................................................................. 70

2.6 SUPPLEMENTARY MATERIAL........................................................... 74

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CHAPTER 3: POLYMORPHISMS IN TOLL-LIKE RECEPTOR (TLR)

GENES INFLUENCE SUSCEPTIBILITY AND

SEVERITY OF ACUTE ASTHMA IN CHILDREN............. 75

3.1 ABSTRACT................................................................................................ 76

3.2 INTRODUCTION...................................................................................... 77


3.4 RESULTS.................................................................................................... 84

3.5 DISCUSSION.............................................................................................. 93


CHAPTER 4: POLYMORPHISMS IN GENES INVOLVED IN THE

RIG-1-LIKE RECEPTOR PATHWAY OF INNATE

IMMUNE RESPONSE TO RHINOVIRUS AFFECT

SUSCEPTIBILITY AND SEVERITY OF ACUTE

ASTHMA IN CHILDREN........................................................ 101

4.1 ABSTRACT................................................................................................ 102

4.2 INTRODUCTION...................................................................................... 104

4.3 METHODS................................................................................................. 106

4.4 RESULTS.................................................................................................... 111

4.5 DISCUSSION.............................................................................................. 119


CHAPTER 5: GENOTYPE-DRIVEN EXPRESSION OF NOVEL

GENES INVOLVED IN CHILDHOOD ACUTE

ASTHMA, IDENTIFIED BY MICRO-ARRAY..................... 127

5.1 ABSTRACT................................................................................................ 128

5.2 INTRODUCTION...................................................................................... 130


5.4 RESULTS.................................................................................................... 139

5.5 DISCUSSION.............................................................................................. 152


xv

CHAPTER 6: GENOTYPES WITHIN STEROID RESPONSIVE GENES

IDENTIFIED BY MICRO-ARRAY ARE ASSOCIATED

WITH CHILDHOOD ACUTE ASTHMA

SUSCEPTIBILITY, SEVERITY AND RESPONSE TO

TREATMENT............................................................................

167

6.1 ABSTRACT................................................................................................ 168

6.2 INTRODUCTION...................................................................................... 170


6.4 RESULTS.................................................................................................... 178

6.5 DISCUSSION.............................................................................................. 193


CHAPTER 7: GENERAL DISCUSSION........................................................ 207

7.1 SUMMARY OF RESULTS……………………………………………... 208

7.2 STUDY LIMITATIONS………………………………………………… 210

7.3 FUTURE DIRECTIONS………………………………………………... 217

7.4 CONCLUSION………………………………………………………….. 219

CHAPTER 8: REFERENCES………………………………………………... 221

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ABBREVIATIONS

A Adenine

ADAM33 Membrane-anchored zinc-dependent metalloproteinase

ADORA3 Adenosine A3 receptor

AdV Adenovirus

AGRF Australian genome research facility

AHR Airway Hyper-responsiveness

AIA Aspirin Induced Asthma

ALOX15B Arachidonate 15-lipoxygenase, type B

cAMP Cyclic adenosine monophosphate

ANOVA Analysis of variance

AP1 Activator protein 1

APC Antigen presenting cell

AR Airway responsiveness

AREG Amphiregulin

ASM Airway smooth muscle

ASTQ Allele-specific transcript quantification

ATF Activating transcription factor

ATP Adenosine triphosphate

ATS American Thoracic Society

2AR 2 adrenergic receptor/adrenoceptor

BAL Bronchoalveolar lavage fluid

BDTv3.1 Big Dye Terminator version 3.1

BoV Bocavirus

bp Base pairs

C Cytosine

cAMP Cyclic AMP

Cardif CARD adaptor inducing IFN

CARD Caspase recruitment domain family

CCL chemokine (C-C motif) ligand

CD Cluster of differentiation

cDNA Complementary DNA

CI Confidence interval

CLAN Clan protein

COPD Chronic obstructive pulmonary disease

CoV Coronavirus

COX Cyclo-oxygenase

xvii

CPE Cytopathic effect

CXCL Chemokine (C-X-C motif) ligand

CXCR Chemokine (C-X-C motif) receptor

DC Dendritic cell

DDX58 DEAD box polypeptide 58

DNA Deoxyribonucleic acid

dNTP Deoxynucleotide triphosphate

dsRNA Double stranded RNA

ECM Extracellular matrix

ED Emergency department

EGF Epidermal growth factor

EGFR Epidermal growth factor receptor

ELISA Enzyme-linked immunosorbent assay

EnV Enterovirus

ER Endoplasmic reticulum

FADD FAS-associated via death domain

FcER1 Fc fragment of IgE, high affinity Ig, receptor for alpha polypeptide

FGF Fibroblast growth factor

FLT3 FMS-related tyrosine kinase 3

FPR2, FPR2A Formyl peptide receptor 2

FPRL1 Formyl peptide receptor-like 1

G Guanine

GM Geometric mean

GMCSF Granulocyte-macrophage colony stimulating factor

GPCR G protein coupled receptor

GWAS Genome-wide association study

HETE Hydroxyeicosatetraenoic acid

HIV Human immunodeficiency virus

HLA-DR Human leukocyte antigen - DR

hMPV Human metapneumovirus

HPETE 15-hydroxy-5,8,11,13-eicosatetraenoic acid

HRV Human rhinovirus

HRVA, B, C HRV groups A, B or C

HWE Hardy-Weinberg Equilibrium

ICAM Intracellular adhesion molecule

IDO Indoleamine 2, 3-dioxygenase

IFIH1 IFN induced with helicase C domain 1

IFI78, IFI-78K Interferon-inducible protein p78 (mouse)

IFN Interferon

xviii

IFNGR1 Interferon gamma receptor 1

Ig Immunoglobulin

I Inhibitor of NF-

I I kinase

IL Interleukin

INDO, IDO indoleamine-pyrrole 2,3 dioxygenase

InfV Influenza virus

IP Interferon γ induced protein

IPS-1 Interferon-beta promoter stimulator 1

IRAK IL-1 receptor-associated kinase

IRF Interferon regulatory factor

ISG Interferon-stimulated gene

ISRE IFN-stimulated response element

ITD Internal tandem duplication

JAK Janus kinase

JM Juxtamembrane

kb kilobases

KIR Killer cell induced immunoglobulin-like receptors

KIR2DL4 Killer cell Ig-like receptor, two domains, long cytoplasmic tail, 4

LD Linkage disequilibrium

LDLR Low-density lipoprotein receptor

LFT Lung function test

LGP2 Laboratory of genetics and physiology 2

LOX Lipo-oxygenase

LPS Lipopolysaccharide

LRR Leucine-rich repeat

LRT Lower respiratory tract

LT Leukotriene

LTB4 Leukotriene B4

LTB4R Leukotriene B4 receptor

LTC4S Leukotriene C4 synthase

M Major allele

m Minor allele

MAF Minor allele frequency

MAPKs Mitogen-activated protein kinases

MAVS Mitochondrial antiviral signalling protein

MCP Monocyte chemotactic protein

Mda5 Melanoma differentiation-associated gene 5

MDI Metered dose inhaler

MIP Macrophage inflammatory protein

xix

MMP Matrix metalloproteinase

MNDA Myeloid cell nuclear differentiation antigen

MS Multiple sclerosis

MS4A4A,

MS4A4, MS4A7

Membrane-spanning 4-domains, subfamily A, member 4, 7

MXA, MX1 Myxovirus (influenza virus) resistance 1

MyD88 Myeloid differentiation primary-response gene 88

NAP1 NF- -activating kinase-associated protein 1

NAP-3 Neutrophil-activating protein 3

NCBI National Centre for Biotechnology Information

NCR Non-coding region

NF-κβ Nuclear factor- κ

NHMRC National Health and Medical Research Council

NIH National Institutes of Health

NK Natural killer

NLR NOD-2 like receptor

NLRC4 NLR family, CARD domain containing 4

NO Nitric oxide

OAS Oligoadenylate synthetase

OR Odds ratio

OX40L, OX-40L OX40 antigen ligand

PAF Platelet activating factor

PAMPs Pathogen associated molecular patterns

PBMC Peripheral blood mononuclear cell

PCAAS Perth childhood acute asthma study

PCR Polymerase chain reaction

pDC Plasmacytoid dendritic cell

PDE Phosphodiesterase

PEF Peak expiratory flow

PF4A, PF4-ALT Platelet factor 4 variant

PF4V1 Platelet factor 4 variant 1

PGE2 Prostaglandin E 2

PI Phosphatidylinositol

PIAF Perth infant asthma follow-up

PIV Parainfluenza virus

PKR IFN-inducible dsRNA-dependent protein kinase

PMH Princess Margaret Hospital for Children

PMN Polymorphonuclear leukocytes

xx

PNS Peripheral nervous system

PPP 5’ triphosphate

PRR Pattern recognition receptors

RT-PCR Reverse transcriptase PCR

qRT-PCR Quantitative RT-PCR

RANTES Regulated upon activation Normal T cell expressed and secreted

RFLP Restriction fragment length polymorphism

RIG-I Retinoic acid inducible gene I

RIP1 receptor-interacting protein 1

RLR RIG-1 like receptors

RMA Respiratory Multicode-Plx Assay

RNA Ribonucleic acid

RSV Respiratory syncytial virus

RTK Receptor tyrosine kinase

RV Rhinovirus

SaO2 Blood oxygen saturation

SAPE Streptavidin phycoerythrin

SD Standard deviation

SLE Systemic lupus erythematosus

SNP Single nucleotide polymorphism

SOCS Suppressors of cytokine signalling

SPARC Secreted protein, acidic, cysteine-rich (osteonectin)

SPT Skin prick test

SSPE Subacute sclerosing panencephalitis

ssRNA single-stranded RNA

STAT Signal transducer and activator of transcription

T1D Type 1 diabetes

T Thymine

TAB TAK1 binding protein

TAK1 Transforming growth factor-beta-activated kinase 1

TANK TRAF-family-member-associated NF-κ activator

TBK1 TANK-binding kinase 1

TGF Transforming growth factor

TF Transcription factor

TH T lymphocyte-helper

THBD Thrombomodulin

THBS Thrombospondin

Th1 or 2 T helper cell type 1 or 2

TICAM1 Toll-like receptor adaptor molecule 1

xxi

TIR Toll/IL-1 receptor

TK Tyrosine kinase

TLR Toll-like receptor

TM Transmembrane

TNF Tumour necrosis factor

TNFAIP6 Tumor necrosis factor, alpha-induced protein 6

TNFR TNF receptor

TNFSF4 Tumor necrosis factor (ligand) superfamily, member 4

TRADD TNFR-associated via death domain

TRAF TNF receptor associated factor

TRIF TIR-domain-containing-adaptor-inducing IFN-β

TRIM Tripartite motif-containing

TSE Target specific extension

TSP Thrombospondin

TSG6, TSG-6 Tumor necrosis factor-stimulated gene 6 protein

ub Ubiquitin

UBE2D2 ubiquitin-conjugating enzyme E2D 2

URT Upper respiratory tract

USA United States of America

UTR Untranslated region

VEGF Vascular endothelial growth factor

VISA Virus-induced signalling adaptor

VP Viral protein

VRI Viral respiratory infection

VSIG4 V-set and immunoglobulin domain containing 4

WHO World Health Organisation

xxii

LIST OF FIGURES

Figure Page

1.1 Airway obstruction in asthma due to bronchoconstriction,

inflammation and mucus hypersecretion.............................................

9

1.2 Interaction between CD4 T cells and B cells important for IgE.......... 12

1.3 Picornaviridae virion........................................................................... 22

1.4 Proposed Pathway of Innate Immune Response to Human

Rhinovirus............................................................................................

26

2.1 Frequency of human rhinovirus (HRV) and other common

respiratory viruses identified in 128 children with an asthma

exacerbation..........................................................................................

69

2.2 Relationship between human rhinovirus (HRV) –C infection and

severity of asthma exacerbation in 128 children..................................

70

3.1 TLR8 SNPs Associated with TLR8 mRNA Levels.............................. 89

3.2 TLR8 rs 4830805 and TLR7 rs5743780 polymorphisms are

Associated with Acute Asthma Severity..............................................

92

S3.1 Haploview Linkage Disequilibrium Plots for TLR3 SNPs

Investigated in Children with Acute Asthma and Non-asthmatic

Children from Perth, Western Australia...............................................

98

S3.2 Haploview Linkage Disequilibrium Plots for TLR7 and TLR8 SNPs



99

4.1 Candidate SNPs Associated with Acute Asthma Susceptibility.......... 114

4.2 IFIH1 rs1990760 SNP Association with MX1 (marker of Type I

IFN) mRNA Level in Convalescence..................................................

116

4.3 Candidate SNPs Associated with Acute Asthma Severity................... 118

S4.1 Haploview Linkage Disequilibrium Plots for DDX58 SNPs



124

xxiii

S4.2 Haploview Linkage Disequilibrium Plots for IFIH1 SNPs



124

S4.3 Haploview Linkage Disequilibrium Plots for MAVS SNPs



125

S4.4 Haploview Linkage Disequilibrium Plots for IL29 SNPs



125

5.1 Novel Candidate Gene SNP Genotype Frequencies in Acute

Asthmatic and Non-Asthmatic Control Children Compared by Chi-

square Analyses....................................................................................

142

5.2 Novel Candidate Gene SNPs Associated with mRNA Expression

Levels...................................................................................................

146

5.3 Novel Candidate Gene SNPs Associated with STAT4 mRNA

Expression Levels...............................................................................

147

5.4 Novel Candidate Gene SNPs Associated with Asthma Exacerbation

Severity Score......................................................................................

150

S5.1 Haploview Linkage Disequilibrium Plots for CXCL5 SNPs



160

S5.2 Haploview Linkage Disequilibrium Plots for PF4V1 SNPs



161

S5.3 Haploview Linkage Disequilibrium Plots for KIR2DL4 SNPs



161

S5.4 Haploview Linkage Disequilibrium Plots for MX1 SNPs



162

S5.5 Haploview Linkage Disequilibrium Plots for SPARC SNPs



162

xxiv

S5.6 Haploview Linkage Disequilibrium Plots for THBS1 SNPs



163

S5.7 Haploview Linkage Disequilibrium Plots for FCER1A SNPs



163

S5.8 Haploview Linkage Disequilibrium Plots for IDO1 SNPs



164

S5.9 Haploview Linkage Disequilibrium Plots for STAT4 SNPs



164

S5.10 Haploview Linkage Disequilibrium Plots for THBD SNPs



165

S5.11 Haploview Linkage Disequilibrium Plots for LTB4R SNPs



165

6.1 NLRC4 rs408813 G/T (A) and TNFSF4 rs1234313 G/A (B)

Genotype Frequencies for Children with Acute Asthma and Non-

asthmatic Children Compared by Chi-squared Analyses.....................

180

6.2 Steroid-responsive Gene SNPs Associated with mRNA Expression... 184

6.3 Steroid-Responsive Gene SNPs Associated with Asthma

Exacerbation Severity...........................................................................

187

6.4 Steroid-Responsive Gene SNPs Associated with Response to Acute

Asthma Treatment................................................................................

191

S6.1 Haploview Linkage Disequilibrium Plots for ALOX15B SNPs



201

S6.2 Haploview Linkage Disequilibrium Plots for FPR2 SNPs



201

xxv

S6.3 Haploview Linkage Disequilibrium Plots for CXCL1 SNPs



202

S6.4 Haploview Linkage Disequilibrium Plots for ADORA3 SNPs



202

S6.5 Haploview Linkage Disequilibrium Plots for FLT3 SNPs



203

S6.6 Haploview Linkage Disequilibrium Plots for MNDA SNPs



203

S6.7 Haploview Linkage Disequilibrium Plots for NLRC4 SNPs



204

S6.8 Haploview Linkage Disequilibrium Plots for VSIG4 SNPs



204

S6.9 Haploview Linkage Disequilibrium Plots for AREG SNPs



205

S6.10 Haploview Linkage Disequilibrium Plots for TNFSF4 SNPs



205

S6.11 Haploview Linkage Disequilibrium Plots for MS4A4A SNPs



206

xxvi

LIST OF TABLES

Table Page

2.1 Population demographics of the Perth Childhood Acute Asthma Study...... 66

2.2 Frequency of common respiratory viruses detected in per-nasal aspirates

from 128 children with an asthma exacerbation............................................

67

2.3 Frequency and type/strain of 112 human rhinoviruses (HRVs) identified in

per-nasal aspirates from 128 children with acute asthma..............................

68

S2.1 Acute asthma severity score at presentation to hospital................................ 74

3.1 Characteristics of Toll-like Receptor Pathway Genes and Single

Nucleotide Polymorphisms (SNPs) Analysed...............................................

83

3.2 Population demographics for the acute asthma cases.................................... 84

3.3 Genotype and Allele Frequencies for SNPs Studied in Acute Asthmatic

and Non-asthmatic Children..........................................................................

86

S3.1 PCR protocol.................................................................................................. 100

4.1 Characteristics of RIG-I-like Receptor Pathway Genes and Single

Nucleotide Polymorphisms (SNPs) Analysed...............................................

110

4.2 Population Demographics for the Acute Asthma Cases................................ 112



113

S4.1 PCR protocol.................................................................................................. 126

5.1 Characteristics of Novel Candidate Genes and Single Nucleotide

Polymorphisms (SNPs) Analysed..................................................................

136



and Non-asthmatic Control Children.............................................................

141

6.1 Characteristics of Steroid Responsive Genes and Single Nucleotide

Polymorphisms (SNPs) Analysed..................................................................

176




181

xxvii

PEER REVIEWED ARTICLES AND PRESENTATIONS

Publications Arising From This Project

Bizzintino, J. and Lee, W.M., Laing, I.A., Vang, F., Pappas, T., Zhang, G., Martin,

A.C., Geelhoed, G.C., Mcminn, P., Goldblatt, J., Gern, J. and LeSouëf, P.N. (2011).

Association between human rhinovirus C and severity of acute asthma in children. Eur

Respir J 37:1037-42 (Awarded the 2010 Louisa Alessandri Memorial Foundation

Publication Award).

Publications During Candidature Related To This Project

Subrata, L. S., Bizzintino, J., Mamessier, E., Bosco, A., McKenna, K. L., Wikstrőm, M.

E., Goldblatt, J., Sly, P. D., Hales, B. J., Thomas, W. R., Laing, I. A., LeSouëf, P. N.

and Holt, P. G. (2009). Interactions between innate antiviral and atopic

immunoinflammatory pathways precipitate and sustain asthma exacerbations in

children. J Immunol 183:2793-800.

Publications During Candidature Unrelated To This Project

Martin, A. C., Zhang, G., Rueter, K., Khoo, S. K., Bizzintino, J., Hayden, C. M.,

Geelhoed, G. C., Goldblatt, J., Laing, I. A., and Le Souëf, P. N. (2008). Beta2-

adrenoceptor polymorphisms predict response to beta2-agonists in children with acute

asthma. The Journal of Asthma 45(5):383.

Bizzintino, J., Khoo, S-K., Zhang, G., Martin, A. C., Rueter, K., Geelhoed, G. C.,

Goldblatt, J., Laing, I. A., LeSouëf, P. N. and Hayden, C. M. (2009). Leukotriene

pathway polymorphisms are associated with altered cysteinyl leukotriene production in

children with acute asthma. Prostaglandins, Leukot & Essent Fatty Acids 81:9-15.

Ali, M., Zhang, G., Thomas, W. R., McLean, C. J., Bizzintino, J., Laing, I. A., Martin,

A.C., Goldblatt, J., LeSouëf, P. N. and Hayden, C. M. (2009). Investigations into the

role of ST2 in acute asthma in children. Tissue Antigens 73:206-12.

Hales, B.J., Martin, A.C., Pearce, L.J., Rueter, K., Zhang, G., Khoo, S-K., Hayden,

C.M., Bizzintino, J., Mcminn, P., Geelhoed, G.C., Lee, W.M., Goldblatt, J., Laing, I.A.,

LeSouëf, P.N. and Thomas, W.R. (2009). Anti-bacterial IgE in the antibody responses

of house dust mite allergic children convalescent from asthma exacerbation. Clin Exp

Allergy 39:1170-8.

Candelaria, P.V., Backer, V., Khoo, S-K., Bizzintino, J., Hayden, C. M., Baynam, G.,

Laing, I. A., Zhang, G., Porsbjerg, C., Goldblatt, J. and LeSouëf, P. N. (2010). The

importance of environment on respiratory genotype/phenotype relationships in the Inuit.

Allergy 65:229-237.

Ng, E. N., Devadason, S. G., Khoo, S-K., Zhang, G., Bizzintino, J., Martin, A.C.,

Goldblatt, J., Laing, I. A., LeSouëf, P. N. and Hayden, C. M. (2010). The role of GSTP1

polymorphisms and tobacco smoke exposure in children with acute asthma. Journal of

Acute Asthma 47(9):1049-56.

Rueter, K., Bizzintino, J., Martin, A., Zhang, G., Hayden, CM., Geelhoed, G., Goldblatt,

J., Laing, IA. and LeSouëf, PN. (2011). Symptomatic viral infection is associated with

impaired response to treatment in children with acute asthma. J Pediatr Aug 18 (Epub).

xxviii

Presentations Arising From This Project

International Conference papers

LeSouëf, P. N., Bizzintino, J., Laing, I. A., Subrata, L. S., Zhang, G., Goldblatt, J. and

Holt, P. G. (2008). Acute asthma in children presenting to an emergency room – role of

infection verses allergy. Collegium Internationale Allergologicum (CIA) 26th

Symposium. Malta. (Poster Presentation).

Bizzintino, J., Lee, W. M., Laing, I. A., Zhang, G., Goldblatt, J., Martin, A. C., Vang,

F., Pappas, T., Gern, J. and LeSouëf, P. N. (2008). Rhinovirus infection in acute asthma

in children presenting to an emergency room. European Respiratory Society (ERS)

Annual Congress. Berlin, Germany. (Poster Presentation).

Lee, W. M., Vang, F., Kim, W., Pappas, T., Gern, J.E., Peiris, M., Bizzintino, J., Laing,

I. A. and LeSouëf, P. N. (2008). Global distribution of new human rhinovirus strains.

10th

International Symposium on Respiratory Viral Infections. Singapore. (Poster

Presentation).

Bizzintino, J., Lee, W. M., Laing, I. A., Zhang, G., Vang, F., Pappas, T., Goldblatt, J.,

Gern, J. and LeSouëf, P. N. (2009). New rhinovirus strains predominate in children with

acute asthma and are associated with more severe exacerbations. European Respiratory

Society (ERS) Annual Congress 2009. Vienna, Austria. (Oral Presentation, Published

Abstract and Conference Press Release).

Bizzintino, J., Laing, I. A., Subrata, L. S., McLean, C. J., Khoo, S-K., Hayden, C. M.,

Goldblatt, J., Holt, P. G. and LeSouëf, P. N. (2009). TLR8 up-regulation during acute

asthma in children, identified by micro-array and confirmed by qRT-PCR, is associated

with TLR8 genotypes. American Thoracic Society (ATS) International Conference

2009. San Diego, California. Am J Respir Crit Care Med 179:A5423. (Poster

Presentation and Published Abstract).

Zhang, G., Khoo, S-K., Bizzintino, J., Martin, A., Goldblatt, J., Laing, I. A. and

LeSouëf, P. N. (2009). Effects of haplotypes form 5 asthma susceptibility genes in

chromosome 5 on total and specific IgE in acute asthmatics. American Thoracic Society

(ATS) International Conference 2009 San Diego, California. Am J Respir Crit Care

Med 179:A2745. (Poster Presentation and Published Abstract).

McLean, C. J., Khoo, S-K., Zhang, G., Laing, I. A., Bizzintino, J., Hayden, C. M.,

Goldblatt, J. and LeSouëf, P. N. (2009). TLR7 and TLR8 Polymorphisms and acute

asthma in children. American Thoracic Society (ATS) International Conference 2009.

San Diego, California. Am J Respir Crit Care Med 179:A4814. (Poster Presentation and

Published Abstract).

Bizzintino, J., Laing, I. A., Subrata, L. S., Zhang, G., Goldblatt, J., Holt, P. G. and

LeSouëf, P. N. (2010). Genotype determined expression of genes differentially

expressed in acute childhood asthma. Collegium Internationale Allergologicum 28th

Symposium. Ischia, Italy. (Poster Presentation).

xxix


LeSouëf, P. N. (2010). Asthma susceptibility is influenced by polymorphisms in genes

involved in childhood acute asthma, identified by micro-array. 2nd International

Congress on Exacerbations of Airway Disease (ICEAD). Miami, Florida. (Oral

Presentation).


LeSouëf, P. N. (2010). Genotype-driven expression of genes involved in childhood

acute asthma, identified by micro-array. American Thoracic Society (ATS) International

Conference 2010. New Orleans, Louisiana. Am J Respir Crit Care Med 181(1): A1317.

(Poster Presentation and Published Abstract).

Cox, D., Martin, A.C., Bizzintino, J., Geelhoed, G. C., Goldblatt, J., LeSouëf, P. N. and

Laing, I. A. (2010). Children with frequent intermittent asthma have the most severe

asthma exacerbations presenting to a tertiary children’s hospital emergency department,

compared to children with infrequent intermittent and persistent asthma. European

Respiratory Symposium (ERS). Barcelona, Spain. (Poster Presentation).

Bizzintino, J., Zhang, G., McLean, C., Khoo, S-K., Subrata, L., Martin, A., Rueter, K.,

Hayden, C., Geelhoed, G., Goldblatt, J., Holt, P., LeSouëf, P. and Laing, I. (2011).

Viral innate immune genotypes associated with susceptibility and severity of childhood

acute asthma. American Thoracic Society (ATS) International Conference 2011.

Denver, Colorado. (Poster Presentation and Published Abstract).

Weeke, LC., Cox, DW., Bizzintino, J., Khoo, S-K., Hayden, CM., Schultz, EN., Zhang,

G., Geelhoed, G., Goldblatt, J., LeSouëf, PN. and Laing, IA. (2011). Human

Rhinovirus Type C In Acute Lower Respiratory Infections In Young Children.

American Thoracic Society (ATS) International Conference 2011. Denver, Colorado.


Cox, D. W., Khoo, S-K., Bizzintino, J., Lee, W. M., Davis, P., Weeke, L. C., Geelhoed,

G. C., Gern, J., Goldblatt, J., LeSouëf, P. N. and Laing, I. A. (2011). Comparison of

Different Types of Nasal Sampling for the Detection of Human Rhinovirus in Children.



Annamalay A, Khoo S-K, Bizzintino J, Chidlow G, Lee W-M, Jacoby P, Moore HC,

Harnett GB, Smith DW, Gern JE, Goldblatt J, Lehmann D, Le Souëf PN and Laing IA

and the Kalgoorlie Otitis Media Research Project (KOMRP) Team (2011). Carriage Of

Human Rhinovirus (HRV)-A Was More Common Than HRVC, In Asymptomatic

Aboriginal And Non-Aboriginal Children Followed From Birth To 2 Years Of Age.



Davis, P., Cox, D. W., Khoo, S-K., Bizzintino, J., Schultz, EN., Lee, W. M., Geelhoed,

G. C., Gern, J., Goldblatt, J., LeSouëf, P. N. and Laing, I. A. (2011). Human

Rhinovirus (HRV)-C is as Common in Children with HRV Who Required Emergency

Treatment for an Acute Respiratory Illness as Symptomatic Sibling Controls. American

Thoracic Society (ATS) International Conference 2011. Denver, Colorado. (Poster

Presentation and Published Abstract).

xxx

National Conference papers

Laing, I. A., Bizzintino, J., Subrata, L. S., McLean, C. J., Khoo, S-K., Hayden, C. M.,

Goldblatt, J., Holt, P. G. and LeSouëf, P. N. (2009). TLR8 up-regulation during acute

asthma in children, identified by micro-array and confirmed by qRT-PCR, is associated

with TLR8 genotypes. Thoracic Society of Australia and New Zealand (TSANZ)

Annual Scientific Meeting 2009. Darwin, Northern Territory, Australia. Respirology.


McLean, C. J., Khoo, S-K., Zhang, G., Laing, I. A., Bizzintino, J., Hayden, C. M.,

Goldblatt, J. and LeSouëf, P. N. (2009). TLR7 and TLR8 Polymorphisms and acute

asthma in children. Thoracic Society of Australia and New Zealand (TSANZ) Annual

Scientific Meeting 2009. Darwin, Northern Territory, Australia. (Poster Presentation).



acute asthma, identified by micro-array. Thoracic Society of Australia and New Zealand

(TSANZ) Annual Scientific Meeting 2010. Brisbane, Queensland Australia.

Respirology. (Poster Presentation and Published Abstract).




acute asthma. Thoracic Society of Australia and New Zealand (TSANZ) Annual

Scientific Meeting 2011. Perth, Western Australia (Oral Presentation).

Local Conference papers

Bizzintino, J. (2008). Rhinovirus causes almost all exacerbations of asthma in children

presenting to an emergency room. Infectious diseases Breakfast seminar. Perth, Western

Australia. (Oral Presentation).


presenting to an emergency room. ICHR Wetlabs Seminar. Perth, Western Australia.

(Oral Presentation).

Bizzintino, J. (2008). Immunogenetics of asthma exacerbations in children. School of

paediatrics and Child Health Seminar. Perth, Western Australia. (Oral Presentation).


presenting to an emergency room. ICHR Postgraduate Forum. Perth, Western Australia.

(Oral Presentation).


presenting to an emergency room. Research and Advances Scientific Meeting. Perth,

Western Australia. (Oral Presentation).


presenting to an emergency room. Thoracic Society of Australia and New Zealand, WA

Branch Annual Conference. Mandurah, Western Australia. (Oral Presentation).

xxxi

Bizzintino, J., Lee, W. M., Laing, I. A., Zhang, G., Vang, F., Pappas, T., Goldblatt, J.,

Gern, J. and LeSouëf, P. N. (2009). New rhinovirus strains predominate in children with

acute asthma and are associated with more severe exacerbations. ICHR Postgraduate

Forum 2009. Perth, Western Australia. (Oral Presentation).



acute asthma, identified by micro-array. Research and Advances Annual Meeting, Perth,

Western Australia. (Oral Presentation).




acute asthma. Child and Adolescent Health Research Symposium, Perth, Western

Australia. (Poster Presentation).

Cox, DW., Khoo, S-K., Bizzintino, J., Goldblatt, J., Laing, IA., and LeSouëf, PN.

(2011). The Prevalence of Human Rhinovirus C is Low in Children From the

Community Without Respiratory Symptoms. Child and Adolescent Health Research

Symposium, Perth, Western Australia. (Oral Presentation).

Scholarships and Awards

2011 Child and Adolescent Health Research Symposium Best Presentation

2011 Thoracic Society of Australia and New Zealand Peter Phelan Paediatric Travel

Grant

2010 Louisa Alessandri Memorial Foundation Scientific Publication Prize

2010 Graduate Research Student Travel Award

2009 Friends of the Institute Travel Grant Award

2009 Postgraduate Research Convocation Travel Award

2009 European Respiratory Society Young Scientist Award

2008 Asthma Foundation of Western Australia PhD Scholarship Supplement

2007 Australian Postgraduate Award (APA)

1

Chapter 1:

Literature Review

2

1.1 Introduction

Asthma, a respiratory condition caused by environmental and genetic factors, is

estimated to affect 300 million people worldwide and is the most common chronic

disease among children (WHO, 2006). As many as one in four primary school children

have asthma in developed countries such as Australia (Janson et al., 1997; Peat et al.,

1994; Robertson et al., 1998). Paediatric asthma represents significant costs to the

individual (morbidity and mortality) and direct and indirect costs to the community

associated with hospitalization and treatment as well as reduced parental working hours

and school attendance (Bousquet et al., 2005). Acute episodes of asthma are important

contributors to these costs and remain a major cause for hospitalisation of children in

developed countries (Asher et al., 1998; Garrett et al., 1988; Ordonez et al., 1998;

Poulos et al., 2005; Wakefield et al., 1997). Common childhood respiratory viral

infections are the most predominant environmental factor associated with asthma

exacerbations. Viruses are detected in up to 85% of childhood asthma exacerbations

and two-thirds of these are rhinovirus (Johnston et al., 1995; Kling et al., 2005). Given

the potential causative role of rhinoviruses in asthma exacerbations, some of the most

important genetic factors influencing asthma may be those involved in the viral genome

and the host’s immune response to rhinovirus. Furthermore, sequence variations that

affect gene function and expression are likely to influence clinical course and

exacerbation severity so that some children experience mild and others severe asthma

attacks. These genetic factors are optimally explored during an asthma attack, since the

immune system could be expected to be most perturbed at this time. These factors

should also be explored in children, since the pathologic features found in asthma may

develop in early childhood. However, the majority of asthma studies have focused on

stable asthma and many of these have been in adults. This project proposes to

investigate the contribution of sequence variations in novel and known host candidate

genes, and in the rhinovirus genome, to acute asthma phenotypes in children.

1.2 Acute asthma

The National Institutes of Health defines asthma as a chronic disease of the airways that

involves inflammation and bronchoconstriction, which obstruct the airways (NIH,

2006). Airway inflammation involves airway tissue influx with numerous infiltrating

inflammatory cells and accompanying fluid, whilst bronchoconstriction is the

constriction of the smooth muscle that surrounds the airways. As a result of this

3

inflammation and bronchoconstriction, asthmatics may suffer recurrent attacks of

breathlessness and wheezing (WHO, 2006).

Acute asthma, also referred to as an asthma attack or exacerbation, is an episode of

worsening asthma symptoms or deterioration in asthmatic state compared with an

individual’s stable state. Acute asthma often requires rescue bronchodilator treatment

and may be severe enough to warrant emergency medical attention. Asthma attacks are

often brought on by triggers (such as allergens, infectious agents or irritants) and/or the

failure to comply with management programs, including medications that largely

control the symptoms of this disease (WHO, 2006).

1.3 Acute asthma symptoms

The clinical manifestations of acute asthma in children include the respiratory

symptoms of wheeze, cough and dyspnoea. Wheezing is a continuous musical

expiratory sound caused by airway obstruction (Weinberger et al., 2007). This raspy or

high-pitched sound can be heard when air passes through narrowed bronchial tubes

(NIH, 2006). Asthmatic wheeze is not to be confused with inspiratory rattling (rales,

crackles or crepitations caused by fluid in the alveoli (Forgacs, 1978; Pasterkamp et al.,

1997) or stridor (a vibratory sound due to upper airway obstruction with tumours,

foreign bodies or infection (Leung et al., 1999), either of which may lead to a

misdiagnosis of asthma (Weinberger et al., 2007). Wheezing in bronchiolar disease

such as asthma is often heard during expiration and can be indicative of a reduction in

peak expiratory flow rate (Shim et al., 1983). Coughing is a common feature of acute

asthma, probably as a function of the body’s attempt to clear sputum or mucus

obstructing the airways. Dyspnoea, also referred to as respiratory distress, is difficulty

in breathing (Thomas, 2005). Signs that a child is dyspnoeic may include shortness of

breath, an increased respiratory rate (number of breaths per minute), inability to speak

in sentences or phrases, cyanosis, (a blue colouration of the skin and mucous

membranes due to oxygen-deprived blood), sweating/cool or clammy skin, nostril

flaring or retractions (the use of accessory muscles; in the sternum, nose, neck and head,

that do not contract during normal breathing, but do contract to actively pull up on the

rib cage during vigorous exercise or significant airway obstruction (Martin et al., 1983;

Thomas, 2005). Although wheeze, cough and dyspnoea are classic symptoms of acute

asthma, they are not definitive.

4

Asthma symptoms are usually associated with widespread airway obstruction that is

extremely variable in nature (Lemanske et al., 2003). This airway obstruction

variability means that the number, degree, frequency and persistence of acute asthma

symptoms can vary greatly in children. The varied frequency and persistence of

symptoms has lead to the characterization of three distinct clinical patterns of asthma

(Guidelines of the National Asthma Council Australia (NACA, 2006)): (1) Infrequent

episodic - individuals who rarely have episodes of acute asthma symptoms (less than

once a month or less than 2 attacks in six months); (2) Frequent episodic - asthmatics

that have frequent asthma symptoms (more than once a month or more than three

attacks in six months) and; (3) Persistent - asthmatics who suffer asthma symptoms on

most days. Whilst symptoms of acute asthma are extremely variable, a common feature

is that symptoms become worse with exercise or at night (WHO, 2006). It is important

to note that any asthmatic child, regardless of their pattern of asthma or previous

number and degree of symptoms, may experience a severe asthma attack that warrants

medical attention.

The main symptom that prompts the need for urgent medical assistance is dyspnoea as a

result of reduced oxygen supply from the lungs to the rest of the body. Children

experiencing dyspnoea with mild asthma attacks usually respond to treatment with

salbutamol and increased inhaled steroid (Volovitz et al., 2001). However, more severe

symptoms (particularly signs of fatigue, drowsiness or confusion) (Roy et al., 2003;

Thomas, 2005), usually requires emergency medical attention.

1.4 Diagnosis of acute asthma

The diagnosis of acute asthma is complicated by the lack of definitive clinical features

and because the major symptoms in presenting children, particularly dyspnoea, may

result from a number of alternative respiratory, cardiac, neurological or other

conditions. Therefore, the diagnosis of acute asthma is based on consideration of the

clinical features and information regarding the history and severity of the presentation.

Physical examination

Several physical signs are evaluated when diagnosing an asthma exacerbation, including

vital signs (body temperature, heart rate, blood pressure and respiratory rate), alertness

and the ability to speak, cough, dyspnoea, respiratory examination (including

auscultation, listening to the internal sounds of the chest with a stethoscope), wheeze

5

and blood oxygen saturation (SaO2). SaO2 is determined with a pulse oximeter, which

is a reliable and non-invasive determinant of arterial oxygen saturation (Burki et al.,

1983; Nolan et al., 2007). Although an asthma diagnosis may be facilitated by lung

function tests, peripheral white blood counts, serum analyses for antibodies to allergens,

analyses for common respiratory viruses and arterial blood gas measurements (to

determine gas exchange levels in the blood related to lung function), testing may be

compromised by a child’s inability to co-operate during an asthma attack and/or the

time required to perform these tests. Therefore, respiratory rate and other signs of

dyspnoea, low oxygen saturation and wheeze are the most commonly used physical

indicators of acute asthma.

Patient history

A history of the presenting condition and other potentially important information

provided by parents may assist medical staff in diagnosing a child with acute asthma.

On presentation to hospital with a suspected asthma attack, a history may include

answers to questions regarding: (i) previous asthma diagnosis, frequency of symptoms

and details of current medication including frequency and compliance; (ii) results of any

previous lung function tests, including airway responsiveness to challenges with

specific stimuli (such as histamine or methacholine) (Yan et al., 1983); (iii) previous ED

visits/ hospitalizations and duration and type of treatment; (iv) circumstances of the

current episode and its aetiology, which includes onset, symptoms, symptom frequency

and duration, treatment administered, allergen exposure, recent respiratory infections

and other recent medications; (v) the child’s general health; and (vi) any family history

of asthma or allergy.

1.5 Acute asthma severity

There are no definitive criteria for assessment of asthma attack severity. However,

reference ranges have been identified for measures indicative of asthma exacerbation

severity, although these are predominantly used for research purposes. The main

measure of acute asthma severity in a research setting is the determination of a clinical

score based on the presence and severity of various common clinical features. Other

tests that can be useful indicators of asthma severity include arterial oxygen saturation

and lung function tests (LFTs).

6

Clinical scores and SaO2

A clinical score is the sum of individually rated clinical signs of asthma severity that are

related to lung function or oxygen deficiency. Such signs include wheezing, accessory

muscle use and dyspnoea (Baughman et al., 1984; Commey et al., 1976; Kerem et al.,

1991; Rahnama'i et al., 2006). Clinical scores are multivariate because the use of a

combination of clinical signs provides more valid information given the complexity of

asthma and because a score is less variable and more reproducible than any of its

components alone (Bishop et al., 1992; van der Windt et al., 1994).

Numerous clinical scores have been used during childhood asthma exacerbations,

including to (i) rate the severity of an asthma exacerbation at a particular time point

(discriminative) (Bishop et al., 1992; Conway et al., 1985; Dawson, 1987a; Dawson,

1987b; Dawson, 1991; Galant et al., 1978; Obata et al., 1992; Oberger et al., 1978;

Rushton, 1982; Wennergren et al., 1986; Wennergren et al., 1992) (ii) predict the

outcome of the exacerbation (predictive) (Bishop et al., 1992; Bishop et al., 1991;

Conway et al., 1985; Dawson, 1987a; Dawson, 1987b; Dawson, 1991; Kerem et al.,

1991; Kerem et al., 1990; Skoner et al., 1987) or; (iii) evaluate the change over time as a

result of an intervention or treatment (evaluative) (Bentur et al., 1992; Bentur et al.,

1990; Guill et al., 1987; Kornberg et al., 1991; Lowell et al., 1987; Mallol et al., 1987a;

Mallol et al., 1987b; Pendergast et al., 1989; Riedler, 1990; Tal et al., 1983; Tal et al.,

1990). Despite differences in purpose, validity, inter-observer agreement and

responsiveness of these scores, a good severity score for asthma according to Bishop et

al should reflect the status of airway physiology, indicate the extent and stability of

treatment response as well as the degree and duration of physical and functional

disability (Bishop et al., 1992).

Whilst there is disagreement on the preference of one score over another, a number of

useful parameters are common to many scoring systems for severity of childhood acute

asthma at presentation including: respiratory rate (increases with severity and can be

adjusted for the child’s age and gender); retractions (which more often involve

sternocleidomastoid muscles in severe asthma than the scalene group of muscles

(Commey et al., 1976); other signs of dyspnoea (including difficulty speaking due to

breathlessness) and; wheeze on auscultation (expiratory, as well as inspiratory in more

severe exacerbations). In addition, retrospectively rated parameters that are common to

numerous clinical scores include: inhaled treatment frequency (the average time

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between bronchodilator treatments); duration of frequent inhalation therapy (for

example the time taken for a child to change from 1-hourly to 2-hourly or 4-hourly

bronchodilator treatments); number of doses of bronchodilator treatment in a given time

period; time to discharge from hospital (number of hours lapsed from presentation to

discharge from hospital); and the total number of ill days (which includes a parental

estimate of how many pre and post-discharge days the child was unwell). Although

clinical scores consist of parameters with clear definitions, they do not replace the need

for continuous physician judgements of a child’s wellbeing and treatment response

(Kerem et al., 1991; McFadden, 1986), but are valuable tools for research when

comparing the severity of asthma exacerbations between children. These clinical scores

are also particularly valuable as they are mostly suitable for children of all ages,

including those aged 2 to 3 years, and they do not require complex or expensive

instrumentation or technical support.

The benefit of using SaO2 (determined by pulse oximetry (Burki et al., 1983; Chapman

et al., 1983)) as a lone marker of childhood acute asthma severity has also been

examined since oxygen saturation may be low (Mihatsch et al., 1990) and hypoxia (a

reduction of oxygen supply to tissues (Thomas, 2005)) is common in asthma

(McFadden et al., 1968). For example, nocturnal arterial oxygen saturation has been

shown to indicate the severity of asthma symptoms that include nocturnal cough, PEF

and daytime LFT measurements in children with acute asthma (Hoskyns et al., 1991).

SaO2 has also been shown to be a predictor for hospitalization and poor outcome for

children with acute asthma in some studies (Geelhoed et al., 1994; Geelhoed et al.,

1988) but not others (Kerem et al., 1990; Mayefsky et al., 1992). Overall, oxygen

saturation may be used as an independent indicator of the severity and outcome of

asthma exacerbations in children.

Lung function tests

Pulmonary function testing is routinely used to determine the extent of airway

obstruction. However, spirometry, including airway responsiveness to challenge or

bronchodilator, airways’ resistance measurement by forced oscillation technique or

interrupter technique (Chowienczyk et al., 1991; van Noord et al., 1989), are used

primarily for the diagnosis of asthma and to measure the severity of airway obstruction

in stable asthma (Burdon et al., 1982; Enright et al., 1994; Orehek et al., 1982; Pratter et

al., 1983; Weinberger et al., 2007). The usefulness of these lung function tests as a

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measure of asthma attack severity in children is limited as: (i) they are largely

dependent on comparison of the child’s lung function when stable; (ii) the

instrumentation must be managed by a trained technician; (iii) they require active

participation which can be difficult for acutely ill or very young children; and (iv) the

measurement of airway obstruction is extremely variable in acute asthma (Enright et al.,

1994; Sly et al., 1990).

1.6 Pathophysiology

1.6.1 Airway obstruction

The pathophysiology and associated symptoms of asthma and exacerbations are

predominantly the result of airway obstruction and can be caused by a number of factors

that include airway inflammation, mucus hypersecretion and bronchoconstriction

(Figure 1.1). Airway obstruction may be intermittent, persistent and/or progressive and

its reversibility (spontaneously or in response to treatment) may be total, partial or it

may be irreversible (Lemanske et al., 2003). This variability exists between patients

and between asthma attacks and is largely responsible for the diversity of symptoms,

response to treatment and exacerbation severities (Lemanske et al., 2003).

Many individuals with asthma have an inappropriate inflammatory immune and/or

bronchoconstriction response to allergen (atopy) or to viral respiratory illness (VRI) (as

recently proposed (Xatzipsalti et al., 2008)), which may be due to intrinsic genetic

susceptibility and/or early life environmental exposures (Braun-Fahrlander et al., 2002;

Illi et al., 2001).

However, not all asthmatics demonstrate these inappropriate responses and rarely

individuals may have these inflammatory or bronchoconstrictive airway responses

without having asthma (Fig 1.1) (Sverrild et al.). Airway hyperresponsiveness to

mannitol and methacholine and exhaled nitric oxide: a random sample population

study).

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Figure 1.1: Airway obstruction in asthma due to bronchoconstriction,

inflammation and mucus hypersecretion (AAAAI, 2005).

Whilst the exact timing and nature of events leading to an asthma attack have not been

elucidated, the mechanisms leading to airway obstruction in asthmatics who suffer an

acute episode have been investigated. Triggers such as allergens or viruses can both set

off an innate immune response and may induce bronchoconstriction, followed by an

adaptive immune response involving T helper cell type (Th) 2 lymphocyte activation,

the overproduction of immunoglobulin (Ig)-E and mast cell activation. Inappropriate

inflammation involving numerous inflammatory cells, such as eosinophils, as well as

mucus hypersecretion causes obstruction of the airways, as does bronchoconstriction

caused by inflammatory mediators (Holt et al., 2009).

Innate immune response

Viruses or bacteria are recognized and captured by innate immune pattern-recognition

receptors (PRRs) on and within epithelial cells, mast cells and antigen presenting cells

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(APCs) (including dendritic cells and macrophages) in the respiratory tract. PRR

activation and signaling activates pro-inflammatory cytokines to instigate and facilitate

an inflammatory response to the pathogen and promote an appropriate adaptive immune

response (Bowie et al., 2008). An appropriate immune response to bacterial and viral

pathogens is thought to involve IFN and IL-12 release from dendritic cells (DCs),

which drive innate immunity and a Th1 type adaptive response, respectively (Reis e

Sousa et al., 1997; Siegal et al., 1999).

Inhaled allergens are detected by DCs located just beneath the airway mucosa, or are

bound to alveolar macrophages that subsequently phagocytose the allergen as part of the

innate immune response (Currie et al., 2000). The result of this allergen capture is the

release of pro-inflammatory mediators, particularly from DCs, that promote antigen

presentation and drive adaptive immune responses (Eisenbarth et al., 2004; Holt et al.,

2004; Lombardi et al., 2009).

Th2 lymphocyte and adaptive immune response

Once APCs have captured exogenous antigen, they internalize and process the antigen

before presenting it to naïve or memory CD4+ T lymphocytes in the lymph nodes.

Naïve CD4+T cells activated by antigen presentation and directed by APC-secreted

cytokines, proliferate and differentiate into regulatory T cells (suppressor T

lymphocytes), memory T cells, or Th lymphocytes, which are characterized by the

profile of produced cytokines (Agrawal et al., 2005; Bharadwaj et al., 2007; Holt et al.,

2004). DCs that produce IL-10 and/or transforming growth factor (TGF) β promote

differentiation into regulatory T cells that can suppress Th1 and Th2 responses with the

expression of Foxp3, IL-10 and/or TGF-β (Bacchetta et al., 2005; Cottrez et al., 2004;

Sakaguchi et al., 1995). Th1 lymphocytes, induced by IL-12-producing APCs, produce

IFN and TNF that activate macrophages and complement pathways to promote

phagocytosis and intracellular pathogen killing as part of the cell-mediated adaptive

immune response (Lombardi et al., 2009). Th2 cells, promoted by IL-4-secreting APCs,

synthesize cytokines that include IL-4, IL-5 and IL-13, which promote IgE synthesis,

leuk

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