Fetal DNA hypermethylation in tight junction pathway is associated with neural tube defects: A genome-wide DNA methylation analysis

Abstract

Neural tube defects (NTDs) are a spectrum of severe congenital malformations of fusion failure of the neural tube during early embryogenesis. Evidence on aberrant DNA methylation in NTD development remains scarce, especially when exposure to environmental pollutant is taken into consideration. DNA methylation profiling was quantified using the Infinium HumanMethylation450 array in neural tissues from 10 NTD cases and 8 non-malformed controls (stage 1). Subsequent validation was performed using a Sequenom MassARRAY system in neural tissues from 20 NTD cases and 20 non-malformed controls (stage 2). Correlation analysis of differentially methylated CpG sites in fetal neural tissues and polycyclic aromatic hydrocarbons concentrations in fetal neural tissues and maternal serum was conducted. Differentially methylated CpG sites of neural tissues were further validated in fetal mice with NTDs induced by benzo(a)pyrene given to pregnant mice. Differentially hypermethylated CpG sites in neural tissues from 17 genes and 6 pathways were identified in stage 1. Subsequently, differentially hypermethylated CpG sites in neural tissues from 6 genes (BDKRB2, CTNNA1, CYFIP2, MMP7, MYH2, and TIAM2) were confirmed in stage 2. Correlation analysis showed that methylated CpG sites in CTNNA1 and MYH2 from NTD cases were positively correlated to polycyclic aromatic hydrocarbon level in fetal neural tissues and maternal serum. The correlation was confirmed in NTD-affected fetal mice that were exposed to benzo(a)pyrene in utero. In conclusion, hypermethylation of the CTNNA1 and MYH2 genes in tight junction pathway is associated with the risk for NTDs, and the DNA methylation aberration may be caused by exposure to benzo(a)pyrene.

Decreased global DNA hydroxymethylation in neural tube defects: Association with polycyclic aromatic hydrocarbons

“…Notably, emerging literatures have shown that 5hmC decrease is associated with human cancer and neural degenerative diseases [12,13]. Although there are several reports on aberrant dynamics of DNA methylation with NTDs [14][15][16][17][18], we are not aware of any studies that have explored the relationship between levels of 5hmC and NTDs, especially with environmental exposure being taken into consideration.…”

Identification and Quantification of Heterogeneously-methylated DNA Fragments Using Epiallele-sensitive Droplet Digital Polymerase Chain Reaction (EAST-ddPCR)

“…In comparison to digital MS-HRM, which allows analysis only in limited compartments like in 96-reaction-well-plates, EAST-ddPCR can be performed in 20,000 up to several 10 6 compartments resulting in significantly higher analytical sensitivities and reproducibility. Sensitivity was determined below 1%, whereas other techniques like Next Generation Sequencing, Pyrosequencing, and SequenomMassArray exhibit sensitivities of 5-10%, also connected to high initial investment and consumable costs for the analysis of heterogeneously methylated epialleles (9,(12)(13)(14).…”

“…Therefore, the analytical sensitivity and precision of this technique is limited. Other techniques, such as next generation sequencing, pyrosequencing and SequenomMassArray, are able to quantify heterogeneously methylated epialleles, but high upfront investment and consumable costs hinder their establishment in routine analytical laboratories (9,(12)(13)(14).…”

Aberrant methylation of Pax3 gene and neural tube defects in association with exposure to polycyclic aromatic hydrocarbons

“…A detailed description on the genome-wide methylation results obtained from Infinium HumanMethylation450 Bead-Chip (HM450K), using DNA isolated from neural tissues from ten NTD cases and eight unrelated non-malformed controls can be found elsewhere [23]. In brief, out of 485,199 CpG sites across the entire genome, 23,294 (4.8%) were differentially methylated between cases and controls.…”