AIMS

To investigate the relationship between maternal smoking habits during pregnancy (i.e. smoking status, sustained smoking/smoking duration, smoking quantity) and DNA methylation levels in cord blood samples from newborn offspring utilising the ALSPAC-ARIES HM450 dataset. This analysis will form part of a meta-analysis across multiple study cohorts.

Overall research question: Is maternal smoking during pregnancy related to CpG site-specific methylation in newborns?

ANALYSIS PLAN

Exposure variable: Four questions have been drawn up to address issues relating to smoking habits throughout pregnancy, timing, dosage and paternal effects. It is acknowledged that not all individual study cohorts will have the relevant data to address all these questions. Hence, each study cohort should address those applicable.

1. Active smoking

a. Sustained active smoking versus no smoking (dichotomous): mothers who smoked during most of the pregnancy/into late pregnancy (2nd/3rd trimester) versus those who did not smoke at all during pregnancy (including those who quit prior to pregnancy).

b. Early pregnancy smoking versus no smoking (dichotomous): mothers who smoked during early pregnancy and quit later versus those who did not smoke at all during pregnancy (including those who quit prior to pregnancy).

c. Ever smoked versus no smoke (dichotomous): mothers who reported smoking at anytime during pregnancy versus those who did not smoke at all during pregnancy (including those who quit prior to pregnancy).

2. Passive smoking

Definition: any indication that mothers were exposed to passive smoking (i.e. partner smoked, other relatives/household members smoked, exposed at home, exposed at work, quantified e.g. greater than 1 hour per day). Perform analysis in non-smokers only, split into passive and non-passive smoking as appropriate (Dichotomous). If possible, perform analyses in the three sets as above.

3. Smoking quantity

Definition: if possible split mothers by 1-9 cigarettes per day, 10+ cigarettes per day, non-smokers (trichotomous). If possible, perform analyses in the three sets as above.

4. Smoking in biological father

If smoking status of biological father is known perform analyses on paternal smoking prior to pregnancy (yes/no, dichotomous).

Outcome variable: DNA methylation utilising the HM450 ARIES data on cord blood samples. If possible, use the raw beta values for all probes i.e. with no normalisations or transformations. Alternatively (or in addition) perform preferred QC and pre-processing analyses as necessary.

Statistical Analysis: Perform robust linear regression with individual CpG site methylation levels as the outcome variable and smoking status as the exposure of interest. Include any potential confounders on a cohort-specific manor. Summary statistics will be provided to Dr Jourbert at NIEHS enabling mixed/random effects modelling in downstream meta-analyses.

Confounders for ALSPAC-ARIES: Definition - any factor associated with the exposure variable (i.e. smoking variable) and plausibly associated with DNA methylation. Assess the potential confounding effects of the following variables and include within the statistical model as necessary: sex, genetic ancestry/ethnical background, social-economical background, maternal age, pre-pregnancy BMI, parity.

Possible Sensitivity analyses: Perform the primary model (sustained vs non-smoke) adjusting for cell composition if possible and adjusting/stratified for preterm birth.

Other stipulations: restrict analyses to singleton births. Do not adjust for gestational age or birth weight in the first instance as these may be on the causal pathway linking smoking, methylation and health outcomes.