MGMT is down-regulated in rats with all-trans retinoic acid-induced spina bifida aperta independently of promoter DNA methylation

The loss of DNA repair genes can cause embryonic teratogenicity and lethality, several DNA repair genes have been reported to be associated with neural tube defects (NTD). O6-methylguanine DNA methyltransferase (MGMT), as one of the DNA repair enzymes has been reported in other congenital malformations, but it is less frequently reported in NTDs. DNA damage was assessed by detecting γ-H2A.X in ATRA-induced SBA rats. Real-time PCR (RT-PCR) was used to examine the mRNA expression of O6-methylguanine DNA methyltransferase (MGMT) in normal and SBA spinal cords. MGMT promoter methylation was analysed using bisulfite sequencing PCR (BSP). Statistical analyses were performed using Student’s t-test and one-way Analysis of Variance (ANOVA). Our data showed that in normal controls, the MGMT mRNA expression was decreased with increasing embryonic days, and decreased dramatically on E11 to E14 (P=0.0483) and reached a minimum on E18 (vs E11, P=0.0031). In SBAs, γ-H2A.X was significantly increased (P=0.0174), and the mRNA expression of MGMT was significantly descreased on E14, E16 and E18 (P=0.0029, 0.0425 and 0.0386, respectively). The BSP results showed that almost all CpG sites in the MGMT promoter remained unmethylated in both SBAs and controls and no significant difference was detected between the two groups in either E14 or E18 embryos. Our results showed that DNA damage occurred in ATRA-induced SBA rat foetuses. The mRNA expression of MGMT is down-regulated in ATRA-induced SBA foetuses, and this down-regulation is independent of promoter DNA methylation.