OBJECTIVES

The objectives of this proposal are:

1) Determine when an independent (of individual socioeconomic characteristics) association between early life area deprivation and lung function, doctor diagnosis of asthma and atopy phenotypes in children at ages 7-9 and 15-17 years emerges.

2) Determine specific area deprivation domains and the individual level life course exposures that mediate an association found in objective 1.

3) Determine whether there is a higher or differential DNA methylation in children of low compared to high parental SEP, and in children living in the worse compared to the least deprived areas (independent of individual level SEP) at 3 points in time (birth, 7, 15-17 years).

4) To investigate whether epigenetic changes found in objective 3 could mediate the association between individual and area deprivation with respiratory-related health outcomes

METHODS

Study participants. ALSPAC is a prospective birth cohort that followed offspring of 14,541 pregnant women resident in the county of Avon with an expected date of delivery between 1st April 1991 and 31st December 1992, from the time of the pregnancy to age 15-17 for this project) (http://www.bristol.ac.uk/alspac/). The study catchment area has a population of 1 million and includes de city of Bristol (0.5 million) with a mixture of rural areas, inner city deprivation, affluent suburbs and moderate sized towns. The cohort of Alspac has been extensively phenotyped and there is a detailed characterization of risk factors and exposures through medical and other administrative records, questionnaires and clinic visits carried out from pregnancy: 4 maternal and 2 partner's questionnaires during pregnancy; 17 mother's, 15 partner's and 23 child-based questionnaires during childhood; with children starting to respond questionnaire form the age of 7y. Since the age of 7y all children were invited to biannual hand-on assessments. DNA has been collected at multiple time points from parents and children. We detail here the measures that are relevant to this project.

Assessment of respiratory health and allergies: The main outcome measures are: 1) Lung function obtained through spirometry at ages 8-9y and 15-17y (FVC, FEV1, FEF25-75); 2) asthma (doctor's diagnosis of asthma at ages 8y and 15y); 3) atopy (Skin Prick test at age 7y and 15y); and, 3) combined asthma and atopy phenotypes which classify children into No asthma and no atopy (reference group), asthma alone, asthma with atopy and atopy alone (see work leading to this project for the socioeconomic patterning at individual level of these phenotypes). Secondary outcomes will include: wheezing phenotypes from birth until age 7 years which classify children based on their patterns of wheezing over time, bronchial reactivity measured with methacoline challenge at age 8y and fractional exhaled nitric oxide (FeNO) concentrations to asses airway inflammation at age 15y.

Assessment of individual socioeconomic position (SEP) and area-level deprivation: These are the main exposures in this project and will be indexed with a variety of indicators aiming to capture all different aspects of this construct67,68. Individual-level SEP is based on education (maternal and paternal), occupational class (maternal and paternal), household income, housing tenure and car access measured during pregnancy, ages 7-8y and 18y. We will analyze specific indicators to evaluate distinct pathways and a composite SEP index, aimed at capturing all different aspects of this construct, obtained with factor analysis. Area-level deprivation measured during 1) Pregnancy: Townsend index of area deprivation derived form 1990 census data at ward level already available in Alspac data (includes indicators of unemployment, overcrowding, car access and home ownership); 2) Two occasions in childhood at ages 8 and 16y based on the Index of multiple deprivation (IMD) 2004 (mostly constructed with 2000 and 2001 data) and 2010 (mostly based with 2008 data) at the Lower layer Super Output Area which has a minimum population of 1000 and a mean population of 1500. The Health domain of the IMD will be excluded to avoid having a marker of health in the exposure variable.

Specific area deprivation domains. The role of specific area attributes will be assessed analysing separately the deprivation domains included in the 2004 and 2010 IMD: Living environment (includes 'indoors' living environment with quality of housing and the 'outdoors' living environment through measures of air quality and road traffic accidents); barriers to housing and services (overcrowding, homelessness, difficulty to access owner occupation; distance to GP, supermarkets, and other services); income domain (including the Income Deprivation Affecting Children Index (Department for Communities and Local Government, 2008), employment domain (considers people of working age who are involuntarily excluded from the world of work, either through unemployment. ill health or family circumstances) (indexed through training and education domain, employment domain), education skills and training domain (which includes two sub-domains: one relating to lack of attainment among children and young people and one relating to lack of qualifications in terms of skills and captures the educational disadvantage of an area); Crime domain (rate of recorded crime for four major crime themes - burglary, theft, criminal damage and violence).

Other area characteristics: Population density (small-area measure of population density, in quintiles); rurality using DEFRA categorisations: 1) urban, rural types (town and fringe, village or hamlet and dispersed); 2)Predominantly urban, predominantly rural, significant rural69; and, length of residence in an area.

Perceived neighbourhood characteristics obtained from maternal self-reported of neighbourhood characteristics will include perceived neighbourhood quality, perceived social problems (crime and disorder), opinion of neighbourhood (good/fairly good/not good/bad) measured during gestation, 2, 5 7, 10 and 17 years.

Assessment of potential confounding, mediating or effect modifying individual level factors: a unique characteristic of Alspac is the extensive information available on a wide range of potential mediating and confounding factors measured since pregnancy and repeatedly throughout the life course of the participating children and their parents. Confounders will include child's age and assay/batch number for epigenetic analysis. All other variables will be considered, a priori, factors that can mediate the association between deprivation and the health outcome. These will include: birth weight; life course exposure to tobacco smoke will be characterized with "in utero" exposure (self-report of maternal smoking during pregnancy), exposure to environmental tobacco smoke (ETS) during childhood through maternal report of child's ETS exposure and blood cotinine levels at age 8y and 15y, child's self-report of smoking initiation at 15y; height and body mass index trajectories throughout childhood; physical activity measured with uniaxial accelerometer at 11, 13, 15 & 17y follow-up; housing characteristics including mould, humidity and temperature in several rooms of the house through maternal and child self-report questionnaire; pet ownership and exposure to pests measured through maternal and child self-report at several points in childhood.

Asssessment of DNA epigenetic changes. DNA has been stored from mothers and children at multiple time points. This is being used by ARIES, a BBSRC-funded resource, to generate epigenomic information based on peripheral blood leucocyte DNA at multiple time points across the lifecourse (http://www.ariesepigenomics.org.uk/). ARIES generates large scale genome-wide DNA methylation analysis of 1000 mother-child pairs measured at pregnancy, birth, age 7y and 15-17y through a Human Illumina 450K array. Illumina 450K data is processed using Illumina's Genome Studio Software. In-assay controls for each stage of the Infinum process are included on the array and analysed for quality prior to data export. Summary statistics of array-wide beta and intensity value are checked to ensure the assay is performing as required. The betas (proportion methylated) for each probe location and probe intensities are then exported. In addition to the measures available through ARIES we seek funding in this project to measure methylation in xxx children in total. To maximize the statistical power to find a difference in the proportion of methylation we will sample xxx children living in the most deprived quartile area and xxx living in the least deprived area at each 3 points in time (see power calculations: pending linking with methylation data). These measures will be obtained following the same ARIES protocol described above.