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PDF file - 663K,Supplementary Figure S1: Confirmation of the quality of RNA in microarray experiments by denaturing gel electrophoresis in 1% agarose. Supplementary Figure S2: Representative pyrograms for PDK4, SPRR2A, SPRR1A, NR1H4 and PYCARD.Supplementary Figure S3: Identification of individual genes and functional gene categories with significant expression changes between BALB/c Mthfr+/- FD and BALB/c Mthfr+/+ CD mice. Supplementary Figure S4: Effect of diet and Mthfr genotype on expression of eight genes in normal intestine of BALB/c mice.Supplementary Figure S5: Correlation of methylation results between quantitative bisulfite-pyrosequencing and the Infinium methylation array. Supplementary Figure S6: Confirmation of DNA methylation differences in normal colon between control subjects and individuals with CRC. Supplementary Table S1: Description of control subjects.Supplementary Table S2: Description of cancer patients.Supplementary Table S3: Primer pairs for qRT-PCR for the 13 genes in Figs S1 and S2, and the internal control, Gapdh.Supplementary Table S4: Taqman primers and probes for qRT-PCR of human genes.Supplementary Table S5: Oligonucleotides used for pyrosequencing (PCR primers and sequencing primers).Supplementary Table S6: List of probe sets showing significant changes comparing Mthfr+/- FD and Mthfr+/+ CD BALB/c mice.Supplementary Table S7: Relative mRNA levels for four genes related to PPAR activation (Bcmo1, Aldh1a1), oxidative stress sensitivity (Sprr2a) or oxidative response (Ugt2b36). Supplementary Table S8: Relative expression of PPAR responsive genes.

Food fortification and increased vitamin intake have led to higher folic acid (FA) consumption by many pregnant women. We showed that FA-supplemented diet in pregnant mice (fivefold higher FA than the recommended level (5xFASD)) led to hyperactivity-like behavior and memory impairment in pups. Disturbed choline/methyl metabolism and altered placental gene expression were identified. The aim of this study was to examine the impact of 5xFASD on the brain at two developmental stages, postnatal day (P) 30 and embryonic day (E) 17.5. Female C57BL/6 mice were fed a control diet or 5xFASD for 1 month before mating. Diets were maintained throughout the pregnancy and lactation until P30 or during pregnancy until E17.5. The 5xFASD led to sex-specific transcription changes in a P30 cerebral cortex and E17.5 cerebrum, with microarrays showing a total of 1003 and 623 changes, respectively. Enhanced mRNA degradation was observed in E17.5 cerebrum. Expression changes of genes involved in neurotransmission, neuronal growth and development, and angiogenesis were verified by qRT-PCR; 12 and 15 genes were verified at P30 and E17.5, respectively. Hippocampal collagen staining suggested decreased vessel density in FASD male embryos. This study provides insight into the mechanisms of neurobehavioral alterations and highlights potential deleterious consequences of moderate folate oversupplementation during pregnancy.