Project 500 CHILD Study
Conditions: Asthma in Children, Allergy in Children, Environmental Exposure, Genetic Predisposition
Sponsor: McMaster University

Title of the Proposal: 
 
“Project 500”  CHILD Study 
 
STUDY METRICS: 
 
Planned number of subjects: 
In total, the CHILD Study recruited 3,629 families 
and 3,350 children are currently being followed.  
We plan to test several key hypotheses by 
analyzing 1 year outcomes in a subset of 500 
children in whom we now have complete datasets 
to that age. 
How long will it take to write and submit protocol 
to IRB/IEC (in months)? 
REB approvals for the full CHILD Study have been 
received at all four recruitment sites – Vancouver, 
Edmonton, Winnipeg, Toronto – and at the National 
Coordinating Centre in Hamilton. All are current. No 
additional ethical approval is required to undertake 
the analyses proposed.  
Projected Start Date (after IRB/IEC and contracting 
are complete): 
Project 500 will commence as soon as funding is 
obtained to perform the biological analyses. 
Expected accrual rate (# of subject/month): 
Recruitment of the full CHILD cohort was completed 
in 2012. 
Projected enrollment timelines: Please specify 
- Date first subject enrolled (mm/yyyy): 
- Date Last subject enrolled (mm/yyyy): 
- Follow-up time (in months): 
The cohort is fully enrolled. 
The 500 children selected for this project have all 
the required data and samples now available. 
Follow-up of all CHILD subjects is planned until 
infants are aged 5 years but this specific project is 
examining outcomes in the first year of life. 
Target time of the first formal analysis (mm/yyyy): 
June 2015 
Target time of the final report (mm/yyyy): 
June 2016 
Time to generate draft manuscript or 
poster/abstract (in months): 
3 months 
Target Publication or Conference (include 
mm/yyyy): 
Data will be presented at national (AllerGen, CSACI) 
and international (ATS, ERS) conferences 2015-16. 
 
 
 
Project 500 Protocol 
 
version: April 18, 2013 

Purpose and rationale for the study: 
 
This proposal relates to the testing of several specific hypotheses in a subset of 500 participants in the 
Canadian Healthy Infant Longitudinal Development (CHILD) Study. These 500 now have complete data 
from the time of recruitment (in pregnancy) to age 1 year. The primary purpose of this proposal is to 
identify risk factors for early allergic outcomes and biomarkers that may predict future disease. These 
500 infants will provide critical preliminary data, not only related to early outcomes, but also to inform 
analytical plans for the full CHILD cohort.   
Overview of the CHILD Study: The CHILD Study was established in 2008 in response to a CIHR RFA, and is 
now firmly established as a multicentre national Canadian cohort study. We have over 3,300 infants in 
follow-up, which will continue until at least age 5 years, and are now embarking on testing some of the 
many hypotheses built into the study, focusing on those related to early outcomes.  
The primary objective of the CHILD Study is to better understand key relationships and interactions among 
the many genetic determinants and environmental exposures associated with the development of 
childhood asthma. Dramatic increases in atopy and asthma over just a few decades suggest new or changing 
environmental exposures, but the developmental origins of atopic disorders remain unresolved. We are 
investigating the root causes of asthma and allergy using a broad-based, longitudinal birth-cohort, which 
recognizes multiple environmental exposures (indoor and outdoor air pollutants, chemicals, infections, 
nutrition, psychosocial environment, drug intake), their timing (pre- and post-natal), and their interactions 
with genetics and epigenetics.  
The overarching hypothesis of the CHILD Study is: The development of childhood asthma is dependent on 
the interactions of genetic predisposition and critical prenatal and early childhood environments 
including nutrition, physical and microbial environments, infections and social environment 
(socioeconomic status and stress). Within this overarching hypothesis, multiple sub-hypotheses will be 
tested within seven interrelated themes, including early life innate immune responses, pre-natal and post-
natal nutrition and intra-partum care, lung function in early childhood and the impact of viral infections, 
early childhood socioeconomic status and life stressors, the impact of the physical environment, specific 
gene-environment interactions, and epigenetic mechanisms. 
  
The objective of Project 500 is to carefully analyze a complete set of multiple clinical and environmental 
questionnaires and selected infant biological samples pertinent to early clinical outcomes (up to age 1 year). 
The primary clinical outcomes are recurrent episodes of wheezing and development of atopy by age 1 
year. Atopy is defined by skin allergen responses. Secondary outcomes include atopic dermatitis or eczema, 
and reported food allergy.  We propose detailed exploration of patterns of clinical illnesses and will 
correlate these with analysis of multiple biomarkers to determine their predictive power.  
 
There is good evidence from longitudinal epidemiological studies and from clinical trials that allergy is both 
a common precursor to childhood asthma and also a predictor of persistence of childhood asthma into 
adulthood. The “atopic march” is a well recognized phenomenon in which children develop atopic 
dermatitis and/or food allergy in early childhood, and then go on to have recurrent wheezing and persistent 
atopic asthma in later childhood and adulthood. Our selection of specific hypotheses for examination in 
Project 500 reflects this phenomenon. 
 
Four specific hypotheses will be tested in “Project 500”: 
 
1. Maternal diet in pregnancy influences food sensitization in infancy; specifically avoidance of foods 
(e.g. peanuts) is associated with intolerance to that food.  
 
Research on diet and interventions to prevent atopic disease have focused on foods with anti-
inflammatory properties (e.g. n-3 fatty acids), antioxidants (vitamin E and zinc), and vitamin D. Recent 
Project 500 Protocol 
 
version: April 18, 2013 

meta-analyses suggest beneficial effects for pre-natal vitamins A, D, and E, zinc, fruits and vegetables, 
and the Mediterranean diet. Nutrients may impact development of asthma through immune 
modulation or the child’s intestinal microbiome. In spite of, or possibly because of, recommendations 
for early life avoidance of specific ‘highly allergenic’ foods including peanut in the 1990s from many 
national expert organizations (e.g. Canadian and British Pediatric Societies, American Academy of 
Pediatrics) there has been an explosion of food allergy, particularly to peanut in most high income 
countries (Miles and Buttriss, Nutrition Bulletin 2010). Intriguingly, studies have shown a very low 
prevalence of peanut allergy in Israel where peanut is commonly introduced in the first year of life 
compared with England where it is seldom introduced before the first birthday. This study accounted 
for a general heritable risk using children from a similar background (Ashkenazi Jewish) in both 
countries. (DuToit et al, JACI 2008).  In the Danish National Birth Cohort maternal peanut (RR: 0.66; 
95% CI: 0.44-0.98) and tree nut (RR: 0.83; 95% CI: 0.70-1.00) intake during pregnancy was associated 
with decreased risk of asthma in the child at 18 months of age. Intriguingly, there was no comment on 
the development of allergy in this cohort. The data we are collecting in the CHILD Study will allow us to 
examine the link between maternal intake of specific foods such as peanut and the outcome of allergy 
to that food in the infant. 
 
2. Low serum 25-hydroxyvitamin D values in infancy are associated with development of early 
childhood wheezing.  
 
Most, but not all, studies have demonstrated a relationship of maternal vitamin D insufficiency with 
wheezing in pre-school children. A meta-analysis of four large cohort studies found that high maternal 
vitamin D intake during pregnancy was protective against wheeze in children. However, a recent 
publication from the ALSPAC cohort shows an association of increased wheeze with increased vitamin 
D levels. One issue may well be polymorphisms of the Vitamin D receptor in the mother (associated 
with vitamin D levels during pregnancy) and in their offspring during the few years of life. The data 
obtained in the CHILD Study will provide the opportunity to confirm or negate the associations between 
levels of vitamin D and wheezing syndromes in early childhood.  
 
3. Sensitization to cow’s milk, egg or peanut, together or separately, at 1 year is a major risk factor for 
wheezing episodes in early infancy.  
 
Sensitization to foods has been associated with an increased risk for asthma (Rhodes et al, J Allergy Clin 
Immunol. 2001;108:720-5 and has recently been included in a modified Asthma Predictive Index (mAPI) 
(NEJM 2006;354:1985-97). The mAPI and an m2API have been validated in a small cohort as good 
predictors for persistent asthma at school age (Chang T et al, J Allergy Clin Immunol: In Practice 2013; 
1:152-156). As yet unpublished data from a Canadian cohort show a substantial increased risk (OR 8-
10) of persistent asthma in children with sensitization to a food in the first year of life (verbal 
communication, Becker A). We will be able, using CHILD Study data, to examine these early life 
sensitization patterns in relation to the occurrence of wheezing episodes, and not only determine 
immediate relationships but also follow the children for several years to validate (or not) the Asthma 
Predictive Index. 
4. Exposures to oxidizing agents in the prenatal period and during the first 3 months of life influence 
the development of atopy and wheeze at 1 year. 
 
Infants raised in Canada spend a large majority of time indoors at home. Multiple indoor air exposures 
have been associated with wheeze and asthma, environmental tobacco smoke (ETS) being the most 
widely recognized.  Oxides of nitrogen, associated with gas cooking and heating, are linked with 
worsening asthma. Innate immune inflammatory response can be activated by common indoor 
exposures, including trichloramines, aldehydes (e.g., formaldehyde) and other volatile organic 
compounds (VOCs) in home furnishings and cleaning products.  
Project 500 Protocol 
 
version: April 18, 2013 

Recent studies suggest that early-life exposure to ambient air pollution may also contribute to the 
development of asthma, with traffic-related air pollution (TRAP) likely playing an important role. TRAP 
represents a complex mixture of pollutants (particulate matter, nitrogen oxides, carbon monoxide, 
organic compounds) in concentrations that are variable over short distances, but that also build-up 
over parts of the city contributing to a variable urban background. Respiratory health risks are generally 
highest among those living close to busy roads with significant truck traffic, but concentration 
variations across urban areas have also been associated with incident asthma.  The effects of TRAP on 
airways disease likely also depend on poorly understood interactions with a range of endogenous or 
exogenous factors such as genetics, co-exposures such as allergens and environmental tobacco smoke, 
and psychosocial stress. The CHILD Study provides an opportunity to study TRAP in four major cities 
with highly variable and well-characterized concentrations, and to examine relationships between 
TRAP, indoor air contaminants, and development of wheeze and atopy in early childhood with the 
intent of following these children through childhood to determine asthma outcomes. 
 
Description: 
Primary Objective(s) and 
Endpoint(s), if different 
from objective(s) 
The primary objective of the full CHILD Study is to better understand key 
relationships and interactions among the many genetic determinants and 
environmental exposures associated with the development of childhood 
asthma, and use this understanding to prevent it and improve its treatment.  
Identification of causal pathways will lead to development and implementation 
of preventative and therapeutic strategies to reduce morbidity and even 
mortality of childhood asthma.  
The primary objective of Project 500 is to analyze multiple clinical and 
environmental questionnaires and selected infant biological samples to 
determine predictors of early clinical outcomes related to allergy and asthma, 
utilizing a subset of children who now have complete data to age 1 year. 
Secondary Objective(s) 
and Endpoint(s), if 
different from 
objective(s) 
The CHILD Study has been developed as a national Canadian resource, 
currently supporting and linking many research projects within the AllerGen 
Network of Centres of Excellence, and supporting several CIHR-funded projects 
including investigation of the role of the human microbiome. The cohort, as 
individuals and through the availability of the detailed database and collection 
of biological samples, will be a platform for not only allergy and asthma related 
research, but potentially for many other investigations of the childhood origins 
of chronic adult diseases, including obesity, cardiovascular and metabolic 
disease. In addition, the availability of clinical data on some 6000 parents of the 
index children will provide fertile material for multiple investigations in adults, 
as well as potentially providing resources for future clinical trials. 
 
Completion of Project 500 will provide sound data on which to base further 
investigations in the full cohort. We propose to explore the relationships 
between early childhood clinical and biomarker outcomes and development of 
overt chronic diseases – asthma and the many other manifestations of allergic 
disease, and cardiovascular and metabolic disorders such as obesity and 
diabetes. We will examine relationships between maternal nutrition and 
infant/childhood health, infant nutrition and childhood health, and interactions 
in multiple fields between genetics and the environment including the role of 
epigenetics. 
 
 
 
Project 500 Protocol 
 
version: April 18, 2013 

Study Design 
CHILD Study Methods: Pregnant women from the general population were 
recruited in the second and third trimester of pregnancy in multiple urban and 
rural centres; they reflect ethnically, environmentally, socioeconomically and 
culturally diverse families with over 25% non-Caucasian parents. Children 
currently range from those recently born up to 5 years old.  
Children are assessed at delivery, at a 3-month home visit, and at 1, 3, and 5 
years of age. Repeated and diverse environmental and health questionnaires 
combined with objective measurements (allergy skin prick tests, lung function) 
detailed home inspection, and a rigorously annotated bank of biological 
samples from parents (venous blood, breast milk), index child (cord and venous 
blood, stool, urine, nasal secretions) and home (dust) provide a robust, 
comprehensive database for testing of multiple interrelated hypotheses.  
Specific methods pertinent to the current analyses: 
Data entry related to events prior to the 1 year assessment have been 
completed as a priority for these 500 children, and data entry for all 1 year 
questionnaires and samples given priority. Questionnaire data will then be 
accessed from HealthDiary, cleaned and assembled by an experienced Data 
Manager. 500 dust samples and 1500 blood samples (mother, cord blood, child 
at 1 year) will be identified and retrieved for analyses. Dust analyses will be 
undertaken at the laboratory of Dr James Scott, University of Toronto, and 
blood analyses at the Clinical Trials and Clinical Research Laboratory, Hamilton. 
Teams of CHILD investigators will analyze the resulting data in collaboration 
with local statisticians and with our Senior Statistician, Dr Wendy Lou, Canada 
Research Chair in Statistical Methods for Health Care, University of Toronto. 
[1] Maternal diet in pregnancy influences food sensitization in infancy, 
specifically avoidance of foods (e.g. peanuts) is associated with intolerance to 
that food.  
Pre- and post-natal nutrition has been collected using maternal and infant/child 
questionnaires including exclusive and non-exclusive breast-feeding, age of 
first supplementation and age of introduction of solid foods. A Food Frequency 
Questionnaire (FFQ) developed by nutritional epidemiologists at the Fred 
Hutchinson Cancer Research Center, based on questionnaires used in US 
studies was modified to reflect Canadian ethnic food choices. Further, the 
database developed by the University of Minnesota Nutrition Data Systems for 
Research for data entry and nutrient analysis was “Canadianized” for use in 
CHILD. This self-administered FFQ asks pregnant mothers to report the 
frequency of consumption and portion size of approximately 175 line items 
during their pregnancy, and was completed by the mother at the time of 
enrollment. At age 3 and 6 months and 1 year, child diet was recorded, 
including exposure to cow’s milk, eggs and nuts.  
[2] Low serum 25-hydroxyvitamin D values in infancy are associated with 
development of early childhood wheezing.  
 
Serum is obtained from children at age 1 year; vitamin D will be measured by 
standard assay methods at the research laboratories at the Hospital for Sick 
Children. Recurrent wheezing will be identified from questionnaires completed 
by mother when the child is 3, 6 and 12 months of age. 
 
Project 500 Protocol 
 
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[3] Sensitization to cow’s milk, egg or peanut, together or separately, at 1 year 
is a major risk factor for wheezing episodes in early infancy.  
 
Epicutaneous testing at 1 year of age is performed to common food allergens 
including cow’s milk, egg and peanut. A positive test (≥2 mm wheal response 
greater than the negative control) will define sensitization regardless of the 
presence of a positive history of an allergic reaction (i.e. history negative skin 
test positive). A definition of food allergy requires the presence of a positive 
test for allergen specific IgE and a positive history of a clinical allergic reaction 
or, in the absence of known exposure to that food, a positive skin test and an 
ex vivo measurement of allergen-specific IgE at a level defined to represent the 
95th percentile or higher for a positive challenge to that food. 
 
[4] Exposures to oxidizing agents in the prenatal period and during the first 3 
months of life influence the development of atopy and wheeze at 1 year. 
 
Data sources: A comprehensive baseline questionnaire (during pregnancy) and 
updates at 3 months, 1, 3, and 5 years of age record residential address, 
housing structure, function, condition, maintenance and cleaning habits, 
presence and use of attached garage, renovations, source and extent of 
dampness, mold growth, new furnishings, appliance and household cleaner 
emissions, presence and type of air conditioning and cleaning, and conditions 
of use.  Questions related to the child’s and families’ time activity include time 
spent in different rooms in the house and indoors vs. outdoors, time in transit, 
mode of transport, frequency/duration of visits to daycare, indoor pools and 
exposure to smoke.  Shorter questionnaires at 6 and 18 months, 2, 2.5, and 4 
years of age focused on child time-activity and major changes including 
renovations. When the infant was 3-4 months old, research staff trained by 
Canada Mortgage and Housing Corporation and the CHILD Environmental 
Working Group visited the home. This enabled measurement of geographical 
coordinates (GPS) for assigning air pollution and traffic-related air pollution 
(TRAP) exposure, evaluation of the structure, function and exposure sources 
within the home with emphasis on cleanliness and cleanability, furnishings, 
ventilation (air conditioning, heating, cooling), potential for moisture build-up, 
microbial and chemical contaminant burdens, basement conditions and 
potential for air exchange with the outside and with attached garages. The 
breadth of exposures characterized will enable development of novel exposure 
indices for integrating multiple oxidative and/or inflammatory exposures to 
better capture total risk posed by the physical environment.  
Indoor Exposures: Questionnaires included animal exposures (dogs, cats, other 
pets) at multiple time points. Dampness (water leaks) and mold were also 
assessed by repeated questionnaires, and visual inspection of the home. Home 
(most used room) and child’s bedroom dust was systematically sampled with a 
specially designed vacuum system, weighed, sieved and stored at -80C. A 
subset of the fine dust has been analyzed for endotoxin and β-glucans to 
support questionnaire validation and application in regards to dampness and 
mold. A range of organic chemicals are also being quantified (e.g., phthalates, 
PAHs) at Environment Canada laboratories. We propose to analyze dust for 
dog, cat, and house dust mite allergens; all children are skin-tested for these 
and other inhalant allergens at 1, 3 and 5 years of age. Urine collected at 3 
Project 500 Protocol 
 
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months and 1 year in a subset of the cohort is currently being analyzed to assess 
exposure to environmental tobacco smoke and phthalate plasticizers. 
Outdoor exposures: We have characterized both large scale patterns and the 
small-scale spatial gradients in TRAP and other combustion-related pollutants 
within each city. With home GPS coordinates, and addresses of locations the 
children frequently visit (e.g., daycare) we can assign a time-weighted air 
pollutant exposure. Land use regression (LUR) models have been developed for 
each city to predict exposure to TRAP. The National Air Pollutant Surveillance 
Program (NAPS) monitoring data and Environment Canada air quality model 
objective analyses are available to capture the larger scale patterns. LUR 
models are consistent across seasons. Time activity data, obtained every 3 
months in year 1, provides weighting to these two levels of outdoor exposures. 
Statistical justification 
for the study design, 
sample size calculation, 
and statistical analysis 
plan 
For Project 500, we have selected 500 children in whom we now have 
complete data to age 1 year. These will comprise approximately 125 children 
from each of the recruitment sites to minimize geographic bias. At age 1 year, 
the dominant clinical outcomes of relevance are wheezing (as defined as a 
positive response to ever wheeze on 3, 6 or 12 month questionnaires), atopic 
dermatitis and reported food allergy, and objective evidence of sensitization to 
food and inhalant allergens assessed by skin testing (≥2mm wheal response to 
epicutaneous testing). We currently have little information on biological data 
at 1 year, as it is rare for normal children to have blood taken at this age. It is 
even less common to have a population of healthy normal children providing 
blood, urine, stool, nasal secretions, and skin allergy tests, to examine 
correlates of biomarkers with these outcomes. Hence power calculations for 
this sample of 500 are difficult, but the planned analyses will provide 
preliminary data which will allow calculations of appropriate sample sizes for 
future analyses in the full cohort.  We anticipate approximately 50 of these 500 
children will have experienced wheezing in the first year of life, and a similar 
number will have developed atopic dermatitis. 
For the full CHILD Study, the primary outcome is physician-confirmed asthma 
at age 5 years, with multiple pre-specified intermediate and secondary 
outcomes. Recruitment and follow-up of over 3,000 families from the general 
population will allow well-powered integrated analyses involving derivation of 
new multi-exposure indices; examining the effect of respiratory infections on 
growth of infant pulmonary function; identification of innate and adaptive 
immune phenotypes in relation to environmental exposures; linkage of 
epigenetic profiles to maternal stress, socioeconomic status, environmental 
exposures and viral infections; and determining impact of nutritional factors 
including maternal diet, breast-feeding, infant diet, transition to table foods 
and the role of probiotics and nutraceuticals on immune function through the 
infant microbiome. The comprehensive longitudinal assessments within CHILD 
allow sophisticated statistical modeling, including longitudinal latent class 
analysis, structural equation modeling and factor analysis to assess specific 
pathways relating genetics, SES, immunity, environment and nutrition to the 
development of allergy and asthma.  
The statistical analyses for Project 500 will include a number of statistical 
approaches, including logistic regression (Agresti, 2012), generalized linear 
models, and generalized linear mixed models (McCulloch et al., 2008).  Both 
Project 500 Protocol 
 
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univariate and multivariate analyses will be employed, and the effects of 
potential confounders will be investigated through multivariable analyses.    
 
All exposure and outcome variables will be first examined, prior to statistical 
modeling. For continuous measures (e.g.  vitamin D intake during pregnancy in 
Hypothesis 1, serum 25-hydroxyvitamin D in infancy in Hypothesis 2, indoor 
concentrations of  endotoxin and β-glucans in Hypothesis 4), the distributions 
(including mean, median, range, variance, etc) will be described and then used 
for determining the appropriateness of possible modifications to measures 
(e.g., log transformation for non-normality, or categorizing values using cut-offs 
based on both statistical and clinical/biological reasons);  for categorical 
measures (e.g., development of wheezing at 1 year in Hypothesis 2, positive 
results for peanut sensitization in skin tests in Hypothesis 3, and development 
of atopy at 1 year in Hypothesis 4), frequencies and percentiles will be 
summarized and used to determine choices of statistical methods (e.g., exact 
methods for sparse data, if applicable).    
 
Specifically, for Hypothesis 1, the association between intolerance to certain 
foods (e.g. peanut) and maternal diet (e.g. exposure to nuts) will be examined 
first using  bivariable logistic regression analysis; here the outcome (binary) 
variable is intolerance (e.g. to peanuts, yes/no), and the explanatory variable is 
diet (e.g. avoidance of peanuts, n-3 fatty acid intake), which can be either a 
binary (yes/no), a categorical (low/medium/high) or a continuous variable.  
When considering the effects of covariates, such as gender and maternal atopy, 
on the association being investigated, multivariable logistic regression analysis 
will be employed, and potential confounders such as ethnicity will be 
accounted for in the regression model. 
 
Similar approaches will be employed in the analyses of the other hypotheses. 
For Hypothesis 2, the outcome variable, development of early childhood 
wheezing (yes/no), will be determined at 1 year, and the measure of serum 25-
hydroxyvitamin D status [25(OH)D] will be examined for its distribution and, if 
necessary, categorized as an ordinal variable (e.g. low/medium/high based on 
statistical and clinical/biological thresholds). Results based on ordinal 
categorization will likely provide an intuitive interpretation of the association 
(e.g. quantifying an n-fold increase in risk for developing childhood wheezing 
when comparing high vs. low 25(OH)D). As a primary exposure variable of 
interest, the distributions of 25(OH)D values will be compared between two 
groups, infants with vs. without wheezing, using nonparametric methods (e.g. 
Wilcoxon rank-sum test) and the t-test (if normally distributed); if the 
distribution exhibits a categorical trend, the chi-square test will be applied to 
evaluate the relationship with the outcome (childhood wheezing).   
 
For Hypothesis 3, the association between the outcome variable, wheezing 
episodes in early infancy (any wheeze to 1 year), and sensitization to foods 
(positive test result) will be examined using regression models for categorical 
data. As described in the previous section “Study Design”, sensitization will be 
defined as a binary variable (positive/negative), and test results for single and 
multiple foods (e.g. sensitivity to egg and peanut) will be considered in the 
analyses, which will employ multiple indicators (so-called “dummy variables” 
for each of the foods tested). To investigate whether there are additive effects 
that combinations of sensitizations to different foods have on the frequency of 
Project 500 Protocol 
 
version: April 18, 2013 

wheezing episodes, the generalized linear models and the generalized linear 
mixed models will be utilized.  
 
When considering the exposures to oxidizing agents under Hypothesis 4, the 
primary outcome variable is the development of atopy and wheeze at 1 year 
(binary, yes/no). The relationships between the outcome and the various 
outdoor and indoor exposures (e.g., home dust mite allergens, continuous 
variable; pet ownership, categorical variable) described in the Study Design will 
be investigated using multivariable logistic regression analyses. The effects of 
other variables, such as genetics and psychosocial stress, will be examined in 
the regression model as covariates.  
 
For all four specific hypotheses, the statistical evaluations will include 
univariate (e.g. distribution of 25(OH)D), bivariate (e.g., association between 
sensitization to peanut and childhood wheezing), and multivariate (e.g., 
relationship between the development of early childhood wheezing and low 
serum 25(OH)D value while controlling for birth weight) analyses.   Variable 
selection, including possible interactions (effect modifications) between 
variables, for the multivariable regression models will be adjudicated based on 
both statistical and clinical significance, and the final model will be determined 
using the Akaike Information Criterion and the Hosmer and Lemeshow 
goodness-of-fit test (Hosmer and Lemeshow, 2000). 
 
All of the approaches mentioned above can be readily implemented using SAS 
(v9.3) and/or R (v2.15). 
 
Study Population , and 
key Inclusion /Exclusion 
criteria 
The 500 children in Project 500 will be representative of our full cohort. Study 
members have been recruited from a general population (i.e., not high-risk for 
asthma and allergy as are many cohorts) from recruitment bases in Vancouver, 
Edmonton, Winnipeg (including a rural site in Manitoba) and Toronto. 
 
Inclusion Criteria  
 
1. Pregnant women aged 18 years and older (19 in Vancouver)  
2. Residence in reasonable proximity to the delivery hospital  
3. Able to read, write, and speak English  
4. Willing to provide informed consent  
5. Willing to consent to cord blood collection for the study  
6. Planning to give birth at a designated recruitment centre participating 
hospital 
7. Infants born at or after 35 weeks  
8. Able to provide address and telephone number and names and 
telephone numbers of two alternate contact individuals  
 
Exclusion Criteria  
 
1. Children with major congenital abnormalities or respiratory distress 
syndrome (RDS)  
2. Expectation of moving away from a recruitment area within year 1  
3. Children of multiple births  
4. Children resulting from in vitro fertilization  
5. Children who will not spend at least 80% of nights in the index home  
Project 500 Protocol 
 
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The families recruited do not include Children in Care, as the essential 
component of the study is determining gene-by-environment effects on 
development of asthma and allergy. This includes the in utero and early home 
environment, and parental genotyping and phenotyping, requiring known birth 
parents and as far as possible a relatively constant environment for optimum 
assessment.  
Treatment information, 
if applicable 
The study is observational, with no planned treatments or interventions. 
Study procedures in 
place to ensure proper 
conduct of the study 
according the principles 
of ICH/GCP 
All procedures and components of the study have been standardized across 
centres, with agreed SOPs for all major components. The Research Project 
Manager makes regular site visits to ensure compliance with SOPs, monitor 
completion of questionnaires and all related data including sample handling 
and labeling. The National Coordinating Centre (NCC) checks all REB 
requirements are met and signed approval given. Data are entered largely at 
the NCC, with procedures for ensuring confidentiality and anonymity of data. 
Study Procedures and 
Assessments 
Quality control procedures are in place for verifying all questionnaire data 
(checking by local research assistants, checking during data entry, queries to 
sites for missing or incomplete data) and biological samples (all divided into 
multiple aliquots to avoid freeze/thaw issues, each aliquot individually 
identified and tracked). Many questionnaires are now answered electronically 
via a secure website, but remain subject to rigorous checking.   
Details of Clinical Safety 
Reporting Processes 
(when applicable provide 
specific risk 
management 
interventions to 
safeguard subjects) 
Not applicable, except that if there should be observation of an obvious danger 
to the child, or instances of child abuse, or other reportable issues noted in the 
family, such would be drawn to the attention of the appropriate authorities. To 
date there have been no such situations identified. 
 
Study Location: 
 
Specify all location(s) where the study will be conducted, incl. Country, name and full address of the institution(s). 
Copy and paste the below fields as needed in order to provide contact details for all participating sites. 
 
Investigator’s name 
Malcolm R Sears on behalf of the CHILD Study 
Name of Institution 
McMaster University / AllerGen NCE 
Address of Institution 
St. Joseph’s Healthcare, 50 Charlton Ave E, Hamilton ON L8N 4A6 
Phone 
905-522-1155 ext 33286 or 33329 
Fax 
905-521-6132 
E-mail 
searsm@mcmaster.ca 
 
Investigator’s name 
Padmaja Subbarao (Toronto site leader) 
Name of Institution 
Hospital for Sick Children 
Project 500 Protocol 
 
version: April 18, 2013 

Address of Institution 
555 University Avenue, Toronto, ON M5G 1X8 
Phone 
416-813-6247 
E-mail 
Padmaja.subbarao@sickkids.ca 
 
Investigator’s name 
Allan Becker (Manitoba site leader) 
Name of Institution 
University of Manitoba 
Address of Institution 
AE101 - 840 Sherbrook Street, Winnipeg, MB R3A 1S1 
Phone 
204-787-2455 
E-mail 
becker@cc.umanitoba.ca 
 
Investigator’s name 
Piushkumar Mandhane (Edmonton site leader) 
Name of Institution 
University of Alberta 
Address of Institution 
2034 RTF 8308 114th Street, Edmonton, AB T6G 2V2 
Phone 
780-407-2753 
E-mail 
mandhane@ualberta.ca 
 
Investigator’s name 
Stuart Turvey (Vancouver site leader) 
Name of Institution 
University of British Columbia 
Address of Institution 
950 West 28th Avenue, Room 371, Vancouver, BC V5Z 4H4 
Phone 
604-875-2345 ext. 5094 
E-mail 
sturvey@cw.bc.ca 
 
 
Investigator’s Name  
Investigator’s Institution  
Andrew Sandford 
University of British Columbia, Vancouver, BC 
Gregory Miller 
Northwestern University, Chicago, IL 
Michael Brauer 
University of British Columbia, Vancouver, BC 
Michael Kobor 
University of British Columbia, Vancouver, BC 
Tobias Kollmann 
University of British Columbia, Vancouver, BC 
Timothy Takaro 
Simon Fraser University, Vancouver, BC 
Anita Kozyrskyj 
University of Alberta, Edmonton, AB 
Project 500 Protocol 
 
version: April 18, 2013 

Dean Befus 
University of Alberta, Edmonton, AB 
Kent HayGlass 
University of Manitoba, Winnipeg, MB 
Felix Ratjen 
The Hospital for Sick Children, Toronto, ON 
Sanja Stanojevic 
The Hospital for Sick Children, Toronto, ON 
Theo Moraes 
The Hospital for Sick Children, Toronto, ON 
Jeffrey Brook 
Environment Canada, Toronto, ON 
James Scott 
University of Toronto, Toronto, ON 
Richard Hegele 
University of Toronto, Toronto, ON 
Wendy Lou 
University of Toronto, Toronto, ON 
Susan Elliott 
University of Waterloo, Waterloo, ON 
Judah Denburg 
McMaster University, Hamilton ON 
Joseph Macri 
McMaster University, Hamilton ON 
Catherine Laprise 
Université du Québec à Chicoutimi, Chicoutimi, PQ 
 
 
 
 
Estimated Study Budget: 
 
The primary costs of Project 500 are related to data and sample analyses. We are collaborating with the 
Canadian Laboratory Initiative in Pediatric Reference Intervals (CALIPER) project to obtain best costs for 
laboratory analyses on our small volume samples.  
 
This application is for $200,000 to be applied as detailed below. 
 
 
 
 
Project 500 Protocol 
 
version: April 18, 2013 

Is monetary funding requested?  
  YES:_____ If yes, give details (per subject costs, cost of study medication, cost of other supplies, other): 
 
This application is for a grant of $200,000 in fiscal year 2012-13 to fund the following components of 
Project 500 of the CHILD study related to analysis of biological samples and clinical data to facilitate 
determination of biomarkers of disease  
 
Data entry of clinical and biological data from 500 children aged 1 year, 3 hours per child 
  for all forms and sample data, data entry clerk rate average $18.00 per hour plus 14% in 
  lieu of benefits (i.e. $20.52):  500 x 3 x $20.52                                                                                         $30,780        
Data manager for 6 months – annual salary $95,000 plus 24% benefits x 0.5                                      $58,900 
 
Retrieval of 1500 blood samples from liquid nitrogen storage @ $2.50 each                                       $  3,750 
Vitamin D, 1500 samples @ $22 per sample                                                                                               $33,000 
Specific IgE (egg, peanut) in 500 blood samples (1 year child ) @ $25 per sample                              $25,000 
Serum ferritin, 1000 samples (mother, child at 1 year) @ 13 per sample                                              $13,000 
Serum folate, 500 samples (mother) @ $23 per sample                                                                           $11,500 
Total serum IgE, 1000 samples (cord blood and 1 year) @$15 per sample                                           $15,000 
Serum IgA (cord blood), 500 samples @ $15                                                                                               $  7,500 
 
Supplies and shipping                                                                                                                                       $  1,570 
 
Total request: 
$200,000 
 
 
Is non-monetary funding requested?  
  NO:____ If yes, give details: 
 
 
Not applicable 
 
 
 
Has funding been received, requested or is intended to seek from any other source?         
  YES:_____ If yes, give details: 
 
The CHILD Study was initiated with parallel grants from CIHR and AllerGen NCE, each committing 
$6,000,000 over 6 years. Further funding has been obtained through grants from Health Canada related to 
sample and data analysis to inform the Chemical Management Plan. Funding from AllerGen has been 
renewed at $2,000,000 per year from April 2012 for 2 years (2012-2014). Grant applications to CIHR and 
other agencies will be pursued for continued follow-up of the cohort and for specific data and sample 
analyses to address the multiple research questions. 
 
Date:   15 April, 2013 
 
 
 
 
Project 500 Protocol 
 
version: April 18, 2013