The invasion cascade exhibited by placental trophoblasts and cancerous cells bears many similarities, and it is attributed to extracellular matrix degradation mediated by matrix metalloproteinases (MMPs). Although proper and controlled invasion by trophoblasts into the maternal uterus is an essential requirement for maintenance of normal pregnancy, any abnormality in this phenomenon results in the development of invasion-related disorders such as gestational trophoblastic diseases (GTDs) and preeclampsia. We studied the epigenetic basis of differential expression of two placental MMPs (MMP2 and MMP9) and tissue inhibitors of metalloproteinases (TIMP2 and TIMP1) during normal gestation and invasion-related disorders, i.e., preeclampsia and GTDs. Our study suggests the association of H3K9/27me3 with differential expression of these MMPs and their inhibitors, which regulate the placental invasion during normal pregnancy, whereas no role of CpG methylation was observed in the differential expression of MMPs/TIMPs. Further, development of GTDs was associated with abnormally higher expression of these MMPs and lower levels of their inhibitors, whereas the reverse trends were observed for MMPs and their TIMPs in case of preeclampsia, in association with abnormal changes in H3K9/27me3. These results suggest the involvement of higher levels of MMPs in an aggressive invasive behavior depicted by GTDs, whereas lower levels of these MMPs in shallow and poor invasive phenotype associated with preeclampsia. Thus, our study shows the significance of a proper balance regulated by histone trimethylation between differential expression of MMPs and their TIMPs for maintaining normal pregnancy and its deregulation as a contributing factor for pathogenesis of invasive disorders during pregnancy.

Placental development is known for its resemblance with tumor development, such as in the expression of oncogenes (c-myc) and telomerase (hTERT). The expression of c-myc and hTERT is up-regulated during early pregnancy and gestational trophoblastic diseases (GTDs). To determine the role of DNA methylation [via methylation-sensitive high resolution melting (MS-HRM)] and histone modifications [via chromatin immunoprecipitation (ChIP assay)] in regulating the differential expression of c-myc and hTERT during normal gestation and their dysregulation during placental disorders, we obtained placental samples from 135 pregnant women, in five groups: normal first, second and third trimester (n = 30 each), pre-eclamptic pregnancy (n = 30) and molar pregnancy (n = 15). Two placental cell lines (JEG-3 and HTR-8/SVneo) and isolated first-trimester cytotrophoblasts were also studied. Quantitative RT-PCR revealed decreased mRNA expression levels of c-myc and hTERT, which were associated with a higher level of H3K9me3 (1.5-fold, P < 0.05) and H3K27me3 (1.9-fold, P < 0.05), respectively, in third-trimester placental villi versus first-trimester villi. A significantly lower level of H3K27me3 in molar placenta was associated with a higher mRNA expression of c-myc and hTERT. The development of pre-eclampsia (PE) was associated with increased methylation (P < 0.001) and H3K27me3 (P < 0.01) at the c-myc promoter and reduced H3K9me3 (P < 0.01) and H3K27me3 (P < 0.05) at the hTERT promoter. Further, mRNA expression of c-myc and hTERT was strongly correlated in molar villi (r = 0.88, P < 0.01) and JEG-3 cells (r = 0.99, P < 0.02). Moreover, on the basis of methylation data, we demonstrate the potential of c-myc as a fetal DNA epigenetic marker for pre-eclamptic pregnancies. Thus we suggest a role for epigenetic mechanisms in regulating differential expression of c-myc and hTERT during placental development and use of the c-myc promoter region as a potential fetal DNA marker in the case of PE.

The circulating cell-free nucleic acids (ccfNAs) are a mixture of single-or doublestranded nucleic acids, released into the blood plasma/serum by different tissues via apoptosis, necrosis, and secretions. Under healthy conditions, ccfNAs originate from the hematopoietic system, whereas under various clinical scenarios, the concomitant tissues release ccfNAs into the bloodstream. These ccfNAs include DNA, RNA, microRNA (miRNA), long non-coding RNA (lncRNA), fetal DNA/RNA, and mitochondrial DNA/RNA, and act as potential biomarkers in various clinical conditions. These are associated with different epigenetic modifications, which show disease-related variations and so finding their role as epigenetic biomarkers in clinical settings. This field has recently emerged as the latest advance in precision medicine because of its clinical relevance in diagnostic, prognostic, and predictive values. DNA methylation detected in ccfDNA has been widely used in personalized clinical diagnosis; furthermore, there is also the emerging role of ccfRNAs like miRNA and lncRNA as epigenetic biomarkers. This review focuses on the novel approaches for exploring ccfNAs as epigenetic biomarkers in personalized clinical diagnosis and prognosis, their potential as therapeutic targets and disease progression monitors, and reveals the tremendous potential that epigenetic biomarkers present to improve precision medicine. We explore the latest techniques for both quantitative and qualitative detection of epigenetic modifications in ccfNAs. The data on epigenetic modifications on ccfNAs are complex and often milieu-specific posing challenges for its understanding. Artificial intelligence and deep networks are the novel approaches for decoding complex data and providing insight into the decision-making in precision medicine.

Imprinted genes occur in discrete clusters that are coordinately regulated by shared DNA elements called Imprinting Control Regions. H19 and Igf2 are linked imprinted genes that play critical roles in development. Loss of imprinting (LOI) at the IGF2/H19 locus on the maternal chromosome is associated with the developmental disorder Beckwith Wiedemann Syndrome (BWS) and with several cancers. Here we use comprehensive genetic and genomic analyses to follow muscle development in a mouse model of BWS to dissect the separate and shared roles for misexpression of Igf2 and H19 in the disease phenotype. We show that LOI results in defects in muscle differentiation and hypertrophy and identify primary downstream targets: Igf2 overexpression results in over-activation of MAPK signaling while loss of H19 lncRNA prevents normal down regulation of p53 activity and therefore results in reduced AKT/mTOR signaling. Moreover, we demonstrate instances where H19 and Igf2 misexpression work separately, cooperatively, and antagonistically to establish the developmental phenotype. This study thus identifies new biochemical roles for the H19 lncRNA and underscores that LOI phenotypes are multigenic so that complex interactions will contribute to disease outcomes.