In utero exposure to maternal smoking is associated with DNA methylation alterations and reduced neuronal content in the developing fetal brain

Background; Intrauterine exposure to maternal smoking is linked to impaired executive function and behavioral problems in the offspring. Maternal smoking is associated with reduced fetal brain growth and smaller volume of cortical grey matter in childhood, indicating that prenatal exposure to tobacco may impact cortical development and manifest as behavioral problems. Cellular development is mediated by changes in epigenetic modifications such as DNA methylation, which can be affected by exposure to tobacco. Results: In this study we sought to ascertain how maternal smoking during pregnancy affects global DNA methylation profiles of the developing dorsolateral prefrontal cortex (DLPFC) during the second trimester of gestation. When DLPFC methylation profiles (Illumina, HM450) of smoking-exposed and unexposed fetuses were compared, no differentially methylated regions (DMRs) passed false discovery correction (FDR ² 0.05). However, the most significant DMRs were hypomethylated CpG Islands within the promoter regions of GNA15 and SDHAP3 of smoking-exposed fetuses. Furthermore, developmental up-regulation of SDHAP3 mRNA was delayed in smoking- exposed fetuses. DMRÕs passing FDR were found by interaction analysis between gestational age and smoking exposure annotated to SYCE3, C21orf56/LSS, SPAG1 and RNU12/POLDIP3. Utilizing established methods to estimate cell proportions by DNA methylation, we found that exposed DLPFC samples contained a lower proportion of neurons in samples from fetuses exposed to maternal smoking. We further showed that nicotine impedes the differentiation of neurons in vitro independent of cell death. Conclusions; We found evidence that intrauterine smoking exposure alters the developmental patterning of DNA methylation and gene expression and is associated with reduced mature neuronal content, effects that are likely driven by nicotine. The Illumina Infinium HM450k Human DNA methylation Beadchip was used to obtain DNA methylation profiles across approximately 480,000 CpGs in 24 fetal samples dissected from the cortical plate in the region that becomes the DLPFC in order to obtain post-migratory NeuN-immunoreactive (NeuN+) neurons in the second trimester of gestation. Samples include 10 fetal cortical sections from mothers with no reported smoking prior to or during pregnancy and 14 fetal cortical sections from mothers that reported smoking prior to and during pregnancy. Genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen) following standard instructions and bisulfite converted (Zymo). Bisulphite converted DNA was amplified, fragmented and hybridised to Illumina Infinium Human Methylation450 Beadchip using standard Illumina protocol