Methylome analysis for spina bifida shows SOX18 hypomethylation as a risk factor with evidence for a complex (epi)genetic interplay to affect neural tube development

Abstract

BackgroundNeural tube defects (NTDs) are severe congenital malformations that arise from failure of neurulation during early embryonic development. The molecular basis underlying most human NTDs still remains largely unknown. Based on the hypothesis that folic acid prevents NTDs by stimulating methylation reactions, DNA methylation changes could play a role in NTDs. We performed a methylome analysis for patients with myelomeningocele (MMC). Using a candidate CpG analysis for HOX genes, a significant association between HOXB7 hypomethylation and MMC was found.MethodsIn the current study, we analyzed leukocyte methylome data of ten patients with MMC and six controls using Illumina Methylation Analyzer and WateRmelon R-packages and performed validation studies using larger MMC and control cohorts with Sequenom EpiTYPER.ResultsThe methylome analysis showed 75 CpGs in 45 genes that are significantly differentially methylated in MMC patients. CpG-specific methylation differences were next replicated for the top six candidate genes ABAT, CNTNAP1, SLC1A6, SNED1, SOX18, and TEPP but only for the SOX18 locus a significant overall hypomethylation was observed (P value = 0.0003). Chemically induced DNA demethylation in HEK cells resulted in SOX18 hypomethylation and increased expression. Injection of sox18 mRNA in zebrafish resulted in abnormal neural tube formation. Quantification of DNA methylation for the SOX18 locus was also determined for five families where parents had normal methylation values compared to significant lower values for both the MMC as their non-affected child. SOX18 methylation studies were performed for a MMC patient with a paternally inherited chromosomal deletion that includes BMP4. The patient showed extreme SOX18 hypomethylation similar to his healthy mother while his father had normal methylation values.ConclusionsThis is the first genome-wide methylation study in leukocytes for patients with NTDs. We report SOX18 as a novel MMC risk gene but our findings also suggest that SOX18 hypomethylation must interplay with environmental and (epi)genetic factors to cause NTDs. Further studies are needed that combine methylome data with next-generation sequencing approaches to unravel NTD etiology.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0272-8) contains supplementary material, which is available to authorized users.

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Finding the genetic mechanisms of folate deficiency and neural tube defects—Leaving no stone unturned

“…A review showed support that a wide variety of environmental factors (e.g., chemical pollutants, diets, temperature changes, and external stresses) can contribute to long‐lasting effects on establishment and maintenance of epigenetic modifications that could thereby influence gene expression and phenotype (Feil & Fraga, ; Price, Peñaherrera, et al, ; Yue, Liu, & He, ). A study of the methylomes in lymphocytes of human SB subjects showed significant overall hypomethylation of SOX18 , a gene not known to associate with NTDs (Rochtus et al, ). This study also showed that addition of sox18 mRNA by injection into zebrafish embryos led to abnormal neural tube closure (Rochtus et al, ).…”

“…A study of the methylomes in lymphocytes of human SB subjects showed significant overall hypomethylation of SOX18 , a gene not known to associate with NTDs (Rochtus et al, ). This study also showed that addition of sox18 mRNA by injection into zebrafish embryos led to abnormal neural tube closure (Rochtus et al, ). In addition, abnormal microRNA profiles were observed in human anencephaly brain tissue (Zhang et al, ) suggesting activity of microRNAs can be modified through post‐transcriptional methylation (Berulava, Rahmann, Rademacher, Klein‐Hitpass, & Horsthemke, ).…”