Research Plan for the CHILD Study
Expectant mothers with singleton pregnancies have been recruited from the general population in Vancouver, Edmonton, Manitoba (Winnipeg and 2 rural sites) and Toronto through pre-natal clinics, physician offices and community programs, usually in their second trimester, and are monitored throughout the remainder of pregnancy.
All children are clinically assessed at delivery, at a 3-month home visit, and at ages 1, 3, and 5 years. Questionnaire data are collected on pre- and post-natal environment, nutrition, stress, health status and medications. Home assessment with dust sample collection at 3 months is complemented by repeated detailed environmental questionnaires from pregnancy to age 5. Anthropometric measures, pulmonary function and viral infections are assessed longitudinally. Biological sampling includes genetic material from the child and parents, cord blood, breast-milk at 3 months, infant peripheral blood at 1 and 5 years, meconium, and viral swabs, urine and stool at multiple time points
CHILD Study Inclusion Criteria:
 pregnant women aged 18 years and older (19 in Vancouver);
 residence in reasonable proximity to the delivery hospital;
 able to read, write, and speak English;
 willing to provide informed consent;
 willing to consent to cord blood collection for the study;
 planning to give birth at a designated recruitment centre participating hospital;
 infants born at or after 35 weeks;
 able to provide name, address and telephone numbers of two alternate contact individuals.
CHILD Study Exclusion Criteria:
 children with major congenital abnormalities or respiratory distress syndrome (RDS);
 expectation of moving away from a recruitment area within 1 year;
 children of multiple births;
 children resulting from in vitro fertilization;
 children who will not spend at least 80% of nights in the index home.
 children born before 35 weeks gestation
Final enrolment was confirmed post-partum. Paternal participation was not mandatory.
VISIT AND DATA CAPTURE PLAN
Specific Research to be Undertaken:
Analysis and Interpretation of Environmental Exposures Across the Cohort:
Development of multiple methods to assess exposures related to the physical environment has been the role of the Environmental Working Group within CHILD. These data will enable CHILD exposure estimates to identify several risk factors, from some already well-recognized (dust mites and cockroaches, traffic-related air pollution) to those that represent newer hypotheses (semi-volatile organic compounds such as phthalates). Questionnaires will be validated, refined, published and synthesized to quantify the exposures and to derive new multi-exposure indices, such as potential oxidant stress burden. In addition to being used to study associations with early outcomes (wheeze, infant pulmonary function test, exhaled nitric oxide (eNO)) and primary endpoints (asthma diagnosis), the exposure data will be shared with partners such as Health and Environment Canada and the CMHC, providing new evidence to inform the establishment of standards and/or guidelines. The Gene x Environment Interactions (GxE) enabling project provides details of studies in CHILD of environmental exposure measurement and modeling technologies, including avoidance strategies.
Analysis and Correlation of Environmental Exposures with wheezing phenotypes in the first three years of life:
The exposures developed for the cohort will be related to clinical phenotypes from questionnaire data. For example, the relationship between the composite score of the oxidant stress burden and infant eNO measurement at birth, three, 12 and 18 months as a marker of airway inflammation will be investigated.
Infant Pulmonary Function Phenotypes and Relation to Viral Infections:
Growth- and respiratory infection-related changes in pulmonary function will be analyzed. It is hypothesized that earlier age of severe infection or multiple early viral infections will predispose children to have decreased growth in airflow measurements. Initial work among three month olds has shown that flow rates are lower than expected for normal controls; using lung clearance index (LCI), a parameter derived from MBW, this team is the first to definitively demonstrate an age-related increase, and the first study to longitudinally follow such a large group of healthy controls. The CHILD Study will provide longitudinal data to determine whether preschool and infant asthmatics have increased LCI compared to healthy controls, and thus identify early asthmatics.
Classification of innate immune phenotypes in relation to environmental exposure and the development of allergy/asthma:
The innate immune system provides the first interface with the microbial world and is essential to initiation of an inflammatory allergic reaction. Toll-like receptors (TLRs) are central for innate recognition of critical pathogen-associated molecular patterns (PAMP). The response of TLRs to PAMPs differs from host to host. Early evidence suggests small differences in TLR sequence (polymorphism) may result in alterations of expression and/or function of the allergic immune response. As the immune system is the earliest final common pathway towards clinically relevant allergy and asthma, a thorough immunological assessment forms the centerpiece of any investigation aimed at correlating differences in environmental exposure with asthma and allergy.
Psychosocial factors and stress in relation to immune function, pulmonary and allergy/asthma outcomes:
The team will examine how the child's psychosocial environment, both pre- and postnatal, relates to the emergence of intermediate phenotypes that foster asthma. On the psychosocial end, the focus will be on the family's economic situation, the quality of the parent-child relationship, and the level of stress present in the home. These risk factors will be related to the newborn's gut microbiota, innate immune response, and cord-blood epigenetic profiles. As the child grows, these risk factors will also be related to trajectories of immune response and pulmonary function, with the idea that high levels of stress will confer risk for a phenotype characterized by excessive cytokine responses to allergens and decreased growth in pulmonary flow measurements. Clinically, these changes will be related to sensitization and the development of wheeze. With the wealth of genomics data in CHILD, the team should also be able to unravel the epigenetic and transcriptional pathways that enable psychosocial factors to "get under the skin." These pathways will suggest novel therapeutic targets
Genetics and Epigenetics in Relation to Allergy / Asthma - Risk and Disease Expression:
The asthma susceptibility gene variants identified in the European Allergy and Asthma Consortium: a Multidisciplinary Study to Identify the Genetics and Environmental Causes of Asthma in the European Community (GABRIEL) cohort accounted for 38-49% of the population-attributable risk, but to date have not been tested in a prospective cohort from the general population. CHILD will enable this critical analysis, avoiding the loss of power due to correction for multiple comparisons that is incurred, e.g., in a genome-wide association study, and applying results to many other clinical outcomes of interest (e.g., food allergy). Whole exome sequencing will be performed on children in the CHILD Study who have a family history of allergy (one or both parents with asthma or another allergic disease) in order to enrich for subjects likely to have rare variants.
The CHILD Study cord and peripheral blood samples will be an invaluable resource for epigenetic studies including indepth epigenetic profiling that will be associated with the wealth of early life phenotypic data as well as with environmental factors such as maternal stress, low socioeconomic status and the composite scores for exposures and viral infections. There have been no large-scale studies in humans of the role of micro RNA (miRNA) in the regulation of gene expression and its relevance to disease pathogenesis. CHILD is uniquely positioned to advance this extremely important and novel area.
Nutritional Factors in Allergy and Asthma:
Using a validated Food Frequency Questionnaire to examine maternal diet during pregnancy and regular questionnaire data about the infants' diets and their transition to table foods, analyses will focus on the role of prenatal food intake previously associated with the development of asthma and allergy. This includes Vitamin D, antioxidants (such as Vitamin E and zinc) and foods with purported anti-inflammatory properties such as omega-3 fatty acids. Novel analyses will examine the role of probiotics, nutraceuticals and alternative medicines (e.g., herbal teas) on atopy and on immune function development. In addition, the role of diet in altering the infant microbiome can be assessed.
Data from the first 991 maternal Food Frequency Questionnaires have revealed a strikingly high prevalence (75-90%) of dietary deficiency for iron and Vitamin D, based on total dietary intakes, when compared with Estimated Average Requirements (EARs) currently recommended by Health Canada for Iron (http://www.hcsc. gc.ca/fn-an/nutrition/reference/ table/ref_elements_tbl-eng.php) and Vitamin D (http://www.hcsc. gc.ca/fn-an/nutrition/reference/table/ ref_vitam_tbl-eng.php). These findings have major public health implications now and in the future for Canadian mothers in pregnancy. CHILD will enable development of a quantitative risk assessment (analogous to dose-response) of these nutritional factors on health outcomes, a major goal of PHAC's Chronic Disease Management and Prevention Strategy
The Infant Microbiome: Microbial Diversity Plays a Significant Role in Atopic Diseases:
Children treated with antibiotics are at greater risk for asthma; probiotics have shown beneficial prophylactic effects for asthma; and gnotobiotic (microbiota-free) animals cannot generate tolerance to antigens. Another example is emerging evidence of breast-milk transmission of the maternal microbiome, potentially impacting the child immune system, and perhaps explaining some of the confusing and conflicting data surrounding the benefit, or lack of benefit, of breastfeeding on the risk of developing allergy and asthma. New experimental approaches will be applied to examining meconium and stool, breast milk and nasal secretions in the CHILD Study. Maternal factors, including mode of delivery (vaginal versus caesarean section), antibiotic use, animal exposure, dietary factors (breast-feeding, introduction of table food) and other environmental influences, all impact the microbiome, both in the gut and airways. These microbiome studies will inform and guide the food and pharmaceutical industries with regard to healthy foods and novel, rational antimicrobial drugs and treatment regimens; public health professionals, regarding formulation of policies involving lifestyle choices for healthy maternal and infant diets and nutrition; and professional (pediatric and obstetric) management guidelines.
BIOLOGICAL SAMPLE COLLECTION PLAN