Blazevic Sofia, Horvaticek Marina, Kesic Maja, Zill Peter, Hranilovic Dubravka, Ivanisevic Marina, Desoye Gernot, Stefulj Jasminka
Department of Biology, Faculty of Science, University of Zagreb, Zagreb, Croatia.
Department of Obstetrics and Gynecology, Clinical Hospital Center Zagreb, Zagreb, Croatia.
PLoS One. 2017 Jun 26;12(6):e0179934. doi: 10.1371/journal.pone.0179934. eCollection 2017.
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 (p<0.05). Placental SLC6A4 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.
我们检验了妊娠期糖尿病（GDM）会改变胎儿血清素转运体基因（SLC6A4）的DNA甲基化模式这一假设，并研究了DNA甲基化对人胎盘组织中SLC6A4表达调控的功能相关性。该研究纳入了50对母婴。18位母亲被诊断为GDM，32位母亲葡萄糖耐量正常（NGT）。所有新生儿出生体重均正常，且均为足月计划性剖宫产出生。分娩后立即从胎盘胎儿侧采集的组织样本中分离出DNA和RNA。使用亚硫酸氢盐处理后的DNA进行PCR扩增并随后进行DNA测序，对SLC6A4远端启动子区域内的7个CpG位点的DNA甲基化进行定量。通过逆转录定量PCR（RT-qPCR）测量SLC6A4 mRNA水平。使用基于标准PCR的方法对功能性SLC6A4多态性（5HTTLPR、STin2、rs25531）进行基因分型。与NGT组相比，GDM组中7个分析位点的平均DNA甲基化水平降低（降低27.1%，p = 0.037），并且在对潜在混杂因素进行调整前后，均与妊娠第24至28周时的母体血浆葡萄糖水平呈负相关（p<0.05）。胎盘SLC6A4 mRNA水平与平均DNA甲基化呈负相关（p = 0.010），而与SLC6A4基因型未发现统计学上的显著关联（p>0.05）。结果表明，胎儿SLC6A4基因的DNA甲基化对孕期母体代谢状态敏感。它们还表明，表观遗传机制在调控人胎盘组织中SLC6A4表达方面比遗传机制起主要作用。需要在更大队列中进行纵向研究以验证这些结果，并确定胎盘SLC6A4变化在多大程度上可能导致暴露于GDM的婴儿的长期结局。
