Epigenome Interactions In Complex Neurogenetic Disorders

Folic acid (FA) supplementation has been shown to decrease the occurrence of neural tube defects (NTDs) in humans. Two allelic mutations in the WNT signaling pathway gene, low density lipoprotein receptor 6 (Lrp6), provide mouse models of neural tube defects (NTDs) with opposing responses to folic acid (FA) supplementation; a high FA diet decreases incidence of NTDs in Crooked Tail (Lrp6Cd/Cd) mice, while increasing NTD occurrence in Lrp6?/? knockout mice. We gathered gene expression (RNA-seq) and DNA methylation (Enhanced-reduced representation bisulfite sequence, or ERRBS) data from two different stages in development of these two strains to investigate the mechanisms of FA rescue, and compared results to WGS and RRBS data from a human cohort of spina bifida patients. Analysis of RNA-seq from embryonic day 9.5 (E9.5) mice found more than 400 genes that were affected by dietary FA levels consistently over all conditions in both strains. Expression of the majority of those genes was suppressed by FA, with an enrichment among genes affecting nuclear processes such as histone and chromatin modification, as well as DNA methylation. Human orthologs of those genes, taken as a set, were significantly enriched for rare (minor allele frequency<0.05) SNPs and indels in the human cohort of 248 patients compared to 380 controls (q-value 3.49E-39, logistic regression). Sixteen (16) of those genes affected by dietary FA levels in mice were also enriched for genetic variants in humans (q-value <0.05, logistic regression) when considered individually, and are thus potentially implicated in the action of FA in humans. Analysis of ERRBS data from postnatal day 2 (P2) animals indicated genome wide effects of both FA supplementation and the two Lrp6 mutant alleles, with 6914 sites being consistently differentially methylated as a result of FA supplementation. The ten (10) genes that were differentially expressed in the RNAseq data according to FA levels in the embryonic animals also showed differential methylation in their promoter regions based on eRRBS at P2. Both Lrp6Cd and Lrp6? alleles were accompanied by pronounced hypermethylation on a control (2ppm) FA diet. On an elevated (10ppm) FA diet, the number of hypermethylated sites increased in the Lrp6+/? mice, while the effect in the Lrp6Cd/+ mice was reversed, inducing a strong hypomethylation signature. Thus, the mouse datasets show epigenetic signatures related to interactions among the Lrp6 locus, genetic background and FA. Upon further validation, the potential orthologus targets that we have identified in humans may allow identification of genetic and epigenetic signatures predicting risk and likely responsiveness to NTD prevention strategies.