Department of Obstetrics and Gynecology, Oakland University William Beaumont School of Medicine, Royal Oak, MI, USA.
Department of Genetics, Cell Biology & Anatomy College of Medicine, University of Nebraska Medical Center, Omaha, NE, USA.
Ultrasound Obstet Gynecol. 2020 Jun;55(6):768-775. doi: 10.1002/uog.20292.
To determine whether the methylation level of cytosine nucleotides in placental DNA can be used to predict tetralogy of Fallot (TOF) and provide insights into the developmental mechanism of this condition.
Tissue sections were obtained from formalin-fixed paraffin-embedded specimens of placental tissue obtained at birth from eight cases with non-chromosomal, non-syndromic TOF and 10 unaffected newborns. The Illumina Infinium HumanMethylation450 BeadChip assay was used to measure cytosine ('CpG' or 'cg') methylation levels at loci throughout the placental genome. Differential methylation was assessed by comparing the β-values (a measure of the extent of cytosine methylation) for individual CpG loci in fetuses with TOF vs in controls. The most discriminating CpG sites were determined based on a preset cut-off of ≥ 2.0-fold change in the methylation level. The predictive accuracy of CpG loci with significant methylation changes for TOF was determined by the area under the receiver-operating-characteristics curve (AUC). A false-discovery-rate (FDR) P-value < 0.05 was used to define a statistically significant difference in the methylation level. Ingenuity Pathway Analysis (IPA) (Qiagen) was used to identify gene pathways that were significantly overexpressed, and thus altered, in TOF cases compared with controls.
We found a total of 165 significantly differentially methylated CpG loci in TOF cases compared with controls, in 165 separate genes. These biomarkers demonstrated from fair to excellent individual predictive accuracy for TOF detection, with AUCs ≥ 0.75 (FDR P-value < 0.001 for all). The following CpG loci (gene) had the highest predictive accuracy: cg05273049 (ARHGAP22; AUC = 1.00; 95% CI, 1.00-1.00), cg02540011 (CDK5; AUC = 0.96; 95% CI, 0.87-1.00), cg08404201 (TRIM27; AUC = 0.95; 95% CI, 0.84-1.00) and cg00687252 (IER3; AUC = 0.95; 95% CI, 0.84-1.00). IPA revealed over-representation (dysregulation) of 14 gene pathways involved in normal cardiac development, including cardiomyocyte differentiation via bone morphogenetic protein receptors, cardiac hypertrophy signaling and role of nuclear factor of activated T cells in cardiac hypertrophy. Cardiac hypertrophy is an important feature of TOF.
Analysis of placental DNA cytosine methylation changes yielded accurate markers for TOF detection and provided mechanistic information on TOF development. Our work appears to confirm the central role of epigenetic changes and of the placenta in the development of TOF. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.
确定胎盘 DNA 中胞嘧啶核苷酸的甲基化水平是否可用于预测法洛四联症（TOF），并深入了解该病的发生机制。
从 8 例非染色体、非综合征性 TOF 患儿和 10 例无异常的新生儿出生时获取的福尔马林固定石蜡包埋胎盘组织的标本中获取组织切片。采用 Illumina Infinium HumanMethylation450 BeadChip 分析测定整个胎盘基因组中胞嘧啶核苷酸（“CpG”或“cg”）甲基化水平。通过比较胎儿 TOF 与对照组之间个体 CpG 基因座的 β 值（衡量胞嘧啶甲基化程度的指标）来评估差异甲基化。基于 2.0 倍变化的预设截止值确定最具区分性的 CpG 位点。CpG 基因座的预测准确率由受试者工作特征曲线（ROC）下的面积（AUC）确定。使用错误发现率（FDR）P 值<0.05 来定义 CpG 基因座的甲基化水平存在统计学差异。使用 IPA（Qiagen）来鉴定与对照组相比 TOF 病例中显著过度表达和改变的基因途径。
与对照组相比，我们在 TOF 病例中发现了总共 165 个差异甲基化 CpG 基因座，涉及 165 个不同的基因。这些生物标志物对 TOF 的检测具有从一般到优秀的个体预测准确性，AUC 均≥0.75（所有 FDR P 值均<0.001）。CpG 基因座（基因）具有最高的预测准确性：cg05273049（ARHGAP22；AUC=1.00；95%CI，1.00-1.00），cg02540011（CDK5；AUC=0.96；95%CI，0.87-1.00），cg08404201（TRIM27；AUC=0.95；95%CI，0.84-1.00）和 cg00687252（IER3；AUC=0.95；95%CI，0.84-1.00）。IPA 显示涉及正常心脏发育的 14 个基因途径的过度表达（失调），包括骨形态发生蛋白受体介导的心肌细胞分化、心脏肥大信号和核因子激活 T 细胞在心脏肥大中的作用。心脏肥大是 TOF 的一个重要特征。
胎盘 DNA 胞嘧啶甲基化变化分析可提供 TOF 检测的准确标志物，并提供 TOF 发生机制的信息。我们的工作似乎证实了表观遗传变化和胎盘在 TOF 发育中的核心作用。版权所有©2019 ISUOG。由 John Wiley & Sons Ltd 出版。
