39: Fetal DNA methylation of autism spectrum disorders (ASD) candidate genes: association with spontaneous preterm birth

Abstract

Autism Spectrum Disorder (ASD) is associated with preterm birth (PTB). Risk factors implicated in ASD are known to result in fetal epigenetic alterations, especially methylation of genes associated with PTB. Our objective was to examine DNA methylation and transcription in four ASD candidate genes in fetal leukocyte and membrane in PTB and pPROM. A systematic literature search for genes implicated in ASD yielded 7 candidate genes (OXTR, SHANK3, BCL2, RORA, EN-2, REELN, MECP2) that were epigenetically modified and linked to ASD. DNA methylation of these 7 genes in fetal leukocyte DNA and their association with PTB were determined from an existing PTB-methylation cohort and four genes (OXTR, SHANK3, BCL2 and RORA) were implicated in both ASD and PTB. This study further evaluated the methylation and its association in fetal membrane DNA from PTB (n=21), pPROM (n=28) and normal term birth (n=43). Quantitative analysis promoter CpG site specific of DNA methylation was performed on four ASD candidate genes using Epitect Methyl qPCR (Qiagen) assay. Expression was also evaluated by QRT-PCR for each gene. ANOVA was used for statistical analysis followed Tukey-test for comparisons between groups. Of the four genes studied, our analysis revealed hypermethylation of oxytocin receptor (OXTR) promoter in PTB compared to term and pPROM (the overall model p=0.05). Methylation of OXTR was significantly higher in PTB than pPROM and term (p=0.03). OXTR mRNA expression was significantly higher in PTB compared to both pPROM and term birth (both p<0.05). Interestingly, in our study, hypermethylation of the OXTR promoter seen in PTB fetal membrane was followed with increased OXTR mRNA expression. We speculate that a novel epigenetic mechanism called hydroxymethylation increases transcriptional activity of genomic regions. Hydroxymethylation studies are underway to further evaluate our hypothesis.