Epigenetic adaptation of the placental serotonin transporter gene (SLC6A4) to gestational diabetes mellitus

We tested the hypothesis that gestational diabetes mellitus (GDM) alters the DNA methylation pattern of the fetal serotonin transporter gene (SLC6A4), and examined the functional relevance of DNA methylation for regulation of the SLC6A4 expression in the human placenta. The study included 50 mother-infant pairs. Eighteen mothers were diagnosed with GDM and 32 had normal glucose tolerance (NGT). All neonates were of normal birth weight and born at term by planned Cesarean section. DNA and RNA were isolated from samples of tissue collected from the fetal side of the placenta immediately after delivery. DNA methylation was quantified at 7 CpG sites within the SLC6A4 distal promoter region using PCR amplification of bisulfite treated DNA and subsequent DNA sequencing. SLC6A4 mRNA levels were measured by reverse transcription—quantitative PCR (RT-qPCR). Functional SLC6A4 polymorphisms (5HTTLPR, STin2, rs25531) were genotyped using standard PCR-based procedures. Average DNA methylation across the 7 analyzed loci was decreased in the GDM as compared to the NGT group (by 27.1%, p = 0.037) and negatively correlated, before and after adjustment for potential confounder/s, with maternal plasma glucose levels at the 24th to 28th week of gestation (pSLC6A4 mRNA levels were inversely correlated with average DNA methylation (p = 0.010) while no statistically significant association was found with the SLC6A4 genotypes (p>0.05). The results suggest that DNA methylation of the fetal SLC6A4 gene is sensitive to the maternal metabolic state in pregnancy. They also indicate a predominant role of epigenetic over genetic mechanisms in the regulation of SLC6A4 expression in the human placenta. Longitudinal studies in larger cohorts are needed to verify these results and determine to which degree placental SLC6A4 changes may contribute to long-term outcomes of infants exposed to GDM.本研究旨在验证妊娠期糖尿病（gestational diabetes mellitus, GDM）可改变胎儿5-羟色胺转运体基因（SLC6A4）的DNA甲基化模式这一假说，并探究DNA甲基化对人胎盘中SLC6A4基因表达调控的功能相关性。本研究共纳入50对母婴，其中18名母亲确诊为GDM，32名母亲糖耐量正常（normal glucose tolerance, NGT）。所有新生儿均为足月正常出生体重儿，且均经计划性剖宫产分娩。分娩后即刻采集胎盘胎儿侧组织样本，从中分离提取DNA与RNA。采用亚硫酸氢盐处理后DNA的PCR扩增结合后续DNA测序的方法，对SLC6A4基因远端启动子区域内的7个CpG位点的DNA甲基化水平进行定量检测。通过逆转录定量PCR（reverse transcription—quantitative PCR, RT-qPCR）测定SLC6A4 mRNA的表达水平。采用标准PCR分型方法对SLC6A4基因的功能性多态位点（5HTTLPR、STin2、rs25531）进行基因分型。与NGT组相比，GDM组7个分析位点的平均DNA甲基化水平降低27.1%（p=0.037）；且无论是否对潜在混杂因素进行校正，该平均甲基化水平均与妊娠24~28周时的母体外周血浆葡萄糖水平呈负相关；SLC6A4 mRNA水平与平均DNA甲基化水平呈负相关（p=0.010），而与SLC6A4基因基因型未检测到统计学显著关联（p>0.05）。研究结果提示，胎儿SLC6A4基因的DNA甲基化水平对妊娠期间母体代谢状态具有敏感性。同时表明，在人胎盘SLC6A4基因的表达调控中，表观遗传机制较遗传机制发挥主导作用。未来需开展更大队列的纵向研究以验证本研究结果，并明确胎盘SLC6A4基因的改变在多大程度上可影响暴露于GDM的婴儿的长期结局。