Placental DNA methylation changes in detection of tetralogy of Fallot

Abstract

Objectives 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. Methods 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. Results 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. Conclusions 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.

Epigenetics and Mechanobiology in Heart Development and Congenital Heart Disease

“…As a result, the cases of CHD that are detected before birth are usually detected well after the heart is malformed. Second, genome-wide analysis has identified distinct pathogenic mutations in only ~10% of cases [10,11,12,13,14,15,16,17,18]. While pathogenic gene mutations are likely involved in the remaining cases, epigenetics and mechanobiology may also cause CHD and certainly exacerbate defects and lead to pathogenic cardiac remodeling.…”

“…Placental tissue is a currently underutilized and non-invasive source of potential biomarkers for the postnatal diagnosis of CHD. One study found 166 CpG loci with differential methylation in placental tissue from TOF [17]. Epigenetic analysis of placentas from patients with VSD revealed 80 CpGs and eight miRNAs that had the potential to accurately detect VSD [170].…”

“…Nodal, bone morphogenic proteins (BMPs), Wnts, Sonic hedgehog, NOTCH, neuregulin, retinoic acid, fibroblast growth factors (FGFs), and other signaling molecules are known to play essential roles in regulating cardiogenesis, primarily by controlling cardiac transcription factors MESP1, GATA4, NKX2.5, HAND1, HAND2, ISL1, and several TBX transcription factors [2,3,4,5,6,7,8,9]. Single mutations in specific morphogens, their target transcription factors, or downstream cardiac genes have been shown to be independently sufficient to cause ~10% of CHD; however, for the majority of cases, the cause remains unknown [10,11,12,13,14,15,16,17,18]. To advance the diagnosis, understanding, and treatment of CHD, the extra-genomic factors critical for heart development need to be uncovered.…”

Placental DNA methylation changes and the early prediction of autism in full-term newborns

“…Statistical and bioinformatic analyses were performed followed by data processing and quality control. A β-value based on the ratio of methylated and unmethylated signal intensities were calculated for each CpG loci using GenomeStudio software as detailed in our prior publications [12,22]. FDR (Benjamini-Hochberg) p-value < 0.05 was considered significant.…”

The Role of Epigenetics in Congenital Heart Disease

“…Interestingly, the use of placental tissue to identify methylation markers in CHD has recently been explored as a potentially useful surrogate for evaluating the development of heart tissue. One study found 165 significantly differentially methylated CpG loci in 8 non-chromosomal, non-syndromic TOF cases compared with 10 unaffected newborns, in 165 separate genes [ 155 ]. Methylation analysis on placentas of 8 term subjects with isolated VSD and 10 unaffected controls revealed 80 CpGs and eight miRNAs that had the potential to accurately detect VSD [ 128 ].…”