Cord Blood Methylation Analysis

For methylation analysis of cord blood, NEST participants were selected based on the availability of recent follow-up data at the time of sample analysis, consent to epigenetic analyses at enrollment, and additional consent to follow-up epigenetic analysis for offspring from ages 3–5 years during follow-up visits. Methylation was assessed in at least one ICR for 1296 participants. Procedures for specimen collection, handling, and methylation are described elsewhere [27 (link),40 (link),71 (link)]. Briefly, DNA for pyrosequencing was extracted from offspring cord-blood buffy coat using Puregene (Qiagen, Germantown, MD) reagents. Pyrosequencing was performed on a Pyromark Q96 Pyrosequencer (Qiagen, Germantown, MD). Primers and PCR conditions are described in detail elsewhere [11 (link),72 (link)]. The methylation fraction for each CpG dinucleotide was calculated using PyroQ CpG Software (Qiagen, Germantown, MD). The methylation fraction was analyzed at multiple CpGs of DMRs (n) in the ICRs of H19 (4), IGF2 (3), MEG3-IG (4), MEG3 (8), MEST (4), PEG3 (10), PLAGL1 (6), NNAT (3), and SGCE/PEG10 (6). Due to quality control methods, minor differences in sample sizes exist depending on the CpG. Note that these regions are considered established ICRs and are referred to as such in the text.