Oragene og 500

Women who responded positively to the invitation letter were sent a recruitment pack (see extended data: Appendix 3
^{12 (link)}). Women who had a live infant were invited to provide a saliva sample from themselves and their baby born whilst participating in the OPPTIMUM study; for those women who had experienced infant loss, an alternative pack was sent with a collection kit for themselves only. The recruitment packs contained a recruitment letter, patient information leaflet, maternal consent form (2 copies), child assent form (if child over 4 years old), instructions for saliva sample collection for mother and maternal saliva sample collection kit (Oragene OG-500, DNA Genotek), a clinical data questionnaire and a postal return kit (postage paid; see extended data
^{12 (link)}). Where appropriate, instructions for saliva sample collection for baby, and an infant saliva sample collection kit (Oragene OG-575, DNA Genotek) was included. If the participant’s baby had died, they were invited to provide a saliva sample from themselves, and an alternative recruitment pack was sent (see extended data: Appendix 3
^{12 (link)}). Women who had not returned the recruitment pack after 6–8 weeks were sent a single reminder letter.

Body weight and fat percentage were assessed at the initial consultation using a SC-331S Total Body Composition Analyzer (TANITA Corp., Tokyo) to measure body weight and estimates fat percentage via Bio-Impedance Analysis. Height was assessed using a wall-mounted stadiometer after removal of shoes. BMI values (kg/m2) were calculated, and BMI z scores and percentiles derived for adolescents based on Center for Disease Control (CDC) growth charts from 2000³⁵ . Adolescents under the 85th percentile were classified as normal-weight, adolescents between the 85th and 95th percentiles as overweight, and adolescents at the 95th percentile or above as obese. During the initial consultation, saliva samples were collected from all adolescents using the Oragene OG500 (DNAGenotek, Ottawa, Canada) saliva collection kits. Adolescents completed the Pubertal Development Scale (PDS)^{36 (link)}. Mothers completed a demographic questionnaire reporting their own and their child’s race/ethnicity.

Genomic DNA was obtained from saliva using Oragene OG-500 (DNA Genotek, Kanata, ON, Canada) saliva kits. Genotyping was performed using custom genotyping arrays (Illumina HumanCoreExome-24 BeadChip), which contain 570 038 genetic variants (Illumina, San Diego, CA, USA). Quality control was implemented in PLINK^{9 (link)} to ensure genotypes did not display ambiguous sex, cryptic relatedness up to third-degree relatives by identity of descent or genotyping completeness <97%. We also removed non-European ethnicity admixture detected as outliers in iterative EIGENSTRAT analyses of an LD-pruned data set.^{10 (link)} SNPs were excluded where the minor allele frequency was <1%, if the call rate <98% or if the χ2-test for Hardy–Weinberg equilibrium had a P-value <1e−04. After quality control, rs322931 genotype information was available for all ninety successfully genotyped individuals.