Decreasing Wapl dosage partially corrects embryonic growth and brain transcriptome phenotypes in Nipbl+/- embryos.

Cohesin rings interact with DNA and modulate the expression of thousands of genes. NIPBL loads cohesin onto chromosomes, and WAPL takes it off. Haploinsufficiency for NIPBL causes a developmental disorder, Cornelia de Lange syndrome (CdLS), that is modeled by Nipbl+/- mice. Mutations in WAPL have not been shown to cause disease or gene expression changes in mammals. Here, we show dysregulation of >1000 genes in WaplΔ/+ embryonic mouse brain. The patterns of dysregulation are highly similar in Wapl and Nipbl heterozygotes, suggesting that Wapl mutations may also cause human disease. Since WAPL and NIPBL have opposite effects on cohesin's association with DNA, we asked whether decreasing Wapl dosage could correct phenotypes seen in Nipbl+/- mice. Gene expression and embryonic growth are partially corrected, but perinatal lethality is not. Our data are consistent with the view that cohesin dynamics play a key role in regulating gene expression.

Folic Acid Levels During Pregnancy Regulate Trophoblast Invasive Behavior and the Possible Development of Preeclampsia.

Background: One of the unique features of placentation is its similarity to tumorigenesis yet being very well regulated. It allows rapid proliferation, migration, and invasion of mononuclear trophoblast cells into the maternal uterus and remodeling the maternal vasculature. This pseudomalignant nature of trophoblastic cells is strictly regulated and its importance becomes evident in abnormal pregnancies that are characterized by aberrant trophoblast proliferation/invasion like preeclampsia. In addition to this, the importance of folic acid supplementation during pregnancy is well documented. We aimed to analyze the molecular and epigenetic regulation of the pseudomalignant nature of placentation via folic acid levels. Methods: Placental tissue samples were collected from different pregnancies in three different gestational stages. We estimated the impact of folic acid levels on global methylation, LINE1 methylation, and expression of DNMTs in all three gestational stages in pregnant women and preeclampsia pregnancies. We also analyzed the effect of folic acid supplementation on trophoblastic invasion using placental derived cells viz, JEG-3 and HTR-8/SVneo cell line and verified the molecular and epigenetic mechanisms involved in this regulation. Results: Development of preeclampsia was observed to be associated with lower folate levels in placental tissue, higher global methylation level, and higher expression of DNMT1and DNMT3A. Folic acid supplementation was found to increase the invasive potential of placental trophoblasts by almost two folds which were associated with the decreased expression of tumor suppressor genes and tissue inhibitors of matrix metalloproteinases; and increased expression of oncogenes, telomerase gene, and matrix metalloproteinases. These folic acid-mediated changes were observed to be regulated by CpG methylation in the case of many genes. Folic acid supplementation was also observed to significantly decrease global methylation in placental trophoblasts related to decreasing expression of DNMT1 and DNMT3A. Conclusion: Lower folic acid levels are associated with preeclampsia development and folic acid supplementation regulates the invasive potential of placental trophoblasts as mediated by various epigenetic changes in the placenta suggesting the protective effect of folic acid against preeclampsia.

Altered dietary ratio of folic acid and vitamin B12 during pregnancy influences the expression of imprinted H19/IGF2 locus in C57BL/6 mice.

Maternal folic acid and vitamin B12 (B12) status during pregnancy influence fetal growth. This study elucidated the effect of altered dietary ratio of folic acid and B12 on the regulation of H19/IGF2 locus in C57BL/6 mice. Female mice were fed diets with nine combinations of folic acid and B12 for 4 weeks. They were mated and the offspring born (F1) were continued on the same diet for 6 weeks post-weaning and were allowed to mate. The placenta and fetal (F2) tissues were collected at day 20 of gestation. H19 overexpression observed under dietary deficiency of folate combined with normal B12 (B12 normal folic acid-deficient, BNFD) was associated with an increased expression of microRNA-675 (miR-675) in maternal and fetal tissues. Insulin-like growth factor 2 (IGF2) expression was decreased under folic acid-deficient conditions combined with normal, deficient or over-supplemented state of B12 (BNFD, BDFD and BOFD) in fetal tissues along with B12 deficiency combined with normal folic acid (BDFN) in the placenta. The altered expression of imprinted genes under folic acid-deficient conditions was related to decreased serum levels of folate and body weight (F1). Hypermethylation observed at the H19 differentially methylated region (DMR) (in BNFD) might be responsible for the decreased expression of IGF2 in female fetal tissues. IGF2 DMR2 was found to be hypomethylated and associated with low serum B12 levels with B12 deficiency in fetal tissues. Results suggest that the altered dietary ratio of folic acid and B12 affects the in utero development of the fetus in association with altered epigenetic regulation of H19/IGF2 locus.

Cardiac pathologies in mouse loss of imprinting models are due to misexpression of H19 long noncoding RNA.

Maternal loss of imprinting (LOI) at the H19/IGF2 locus results in biallelic IGF2 and reduced H19 expression and is associated with Beckwith--Wiedemann syndrome (BWS). We use mouse models for LOI to understand the relative importance of Igf2 and H19 mis-expression in BWS phenotypes. Here we focus on cardiovascular phenotypes and show that neonatal cardiomegaly is exclusively dependent on increased Igf2. Circulating IGF2 binds cardiomyocyte receptors to hyperactivate mTOR signaling, resulting in cellular hyperplasia and hypertrophy. These Igf2-dependent phenotypes are transient: cardiac size returns to normal once Igf2 expression is suppressed postnatally. However, reduced H19 expression is sufficient to cause progressive heart pathologies including fibrosis and reduced ventricular function. In the heart, H19 expression is primarily in endothelial cells (ECs) and regulates EC differentiation both in vivo and in vitro. Finally, we establish novel mouse models to show that cardiac phenotypes depend on H19 lncRNA interactions with Mirlet7 microRNAs.

Circulating Cell-Free Nucleic Acids as Epigenetic Biomarkers in Precision Medicine.

The circulating cell-free nucleic acids (ccfNAs) are a mixture of single- or double-stranded 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.

Temporal expression of genes involved in folate metabolism and transport during placental development, preeclampsia and neural tube defects.

Folate is an essential micronutrient during pregnancy. The differential expression of genes related to folate transport and metabolism during the advancing gestation and pregnancy complications is not well established. Hence, we studied the gene expression of folate metabolism and transport proteins in the placenta with advancing gestation, preeclampsia and neural tube defects (NTD). The expression of folate transporters and enzymes involved in folate metabolism in the placenta with advancing gestation and pregnancy-related disorders were studied by 2-step RT-PCR. Folate levels were estimated by microbiological assay using Lactobacillus casei. Significant changes in levels of placental folate metabolizing enzymes were found in both physiological and pathological pregnancies during advancing gestation. Expression of methyltetrahydrofolate reductase (MTHFR) (p < 0.001) and cystathionine-ß-synthase (CBS) (p < 0.001) was decreased while that of methionine synthase (MS) (p < 0.001) was increased with advancing gestation. A much-reduced expression of MTHFR (p < 0.01) and an abnormally high expression of methionine synthase reductase (p < 0.001) were observed in the NTD group. In NTDs, there was an adaptive up-regulation of folate transporters mainly reduced folate carrier (p < 0.001) and folate receptor alpha (p < 0.001). MTHFR expression showed a strong positive correlation (r = 0.96, p < 0.01) with folate levels in placenta. Pregnant women with preeclampsia had low expression of MS (p < 0.01) in association with low folate levels. Placental folate metabolizing enzymes exhibited a differential pattern during advancing gestation. Deficient folate status in association with alteration in expression of enzymes involved in folate metabolism might be associated with pregnancy complications such as preeclampsia and NTDs.

Modulation of dietary folate with age confers selective hepatocellular epigenetic imprints through DNA methylation.

The present study has been designed to determine the effect of folate modulation (deficiency/supplementation) with aging on the promoter methylation of tumor suppressor and proto-oncogenes to understand the underlying mechanism of epigenetic alterations. Folate deficiency was induced for 3 and 5 months in weanling, young and adult groups, and after 3 months of folate deficiency, they were repleted with physiological folate (2 mg/kg diet) and folate oversupplementation (8 mg/kg diet) for another 2 months. The methylation facet in the present study revealed that the combined effect of folate deficiency and aging decreased the methylation index. Folate deficiency with age resulted in the up-regulation of proto-oncogenes (C-MYC and C-JUN) and cell cycle regulator gene Cyclin E as a result of promoter hypomethylation. However, in case of tumor suppressor genes (p53, p15ink4b and p16ink4a), the expression levels were found to be decreased at transcriptional level due to promoter hypermethylation. Upon repletion with physiological folate and folate oversupplementation, we found down-regulation of proto-oncogenes and up-regulation of tumor suppressor genes as a result of promoter hypermethylation and hypomethylation, respectively. Deregulation of these important genes due to folate deficiency may contribute toward the pathogenesis at cellular level.

Loss of imprinting mutations define both distinct and overlapping roles for misexpression of IGF2 and of H19 lncRNA.

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.

Epigenetic modifications at DMRs of placental genes are subjected to variations in normal gestation, pathological conditions and folate supplementation.

Invasive placentation and cancer development shares many similar molecular and epigenetic pathways. Paternally expressed, growth promoting genes (SNRPN, PEG10 and MEST) which are known to play crucial role in tumorogenesis, are not well studied during placentation. This study reports for the first time of the impact of gestational-age, pathological conditions and folic acid supplementation on dynamic nature of DNA and histone methylation present at their differentially methylated regions (DMRs). Here, we reported the association between low DNA methylation/H3K27me3 and higher expression of SNRPN, PEG10 and MEST in highly proliferating normal early gestational placenta. Molar and preeclamptic placental villi, exhibited aberrant changes in methylation levels at DMRs of these genes, leading to higher and lower expression of these genes, respectively, in reference to their respective control groups. Moreover, folate supplementation could induce gene specific changes in mRNA expression in placental cell lines. Further, MEST and SNRPN DMRs were observed to show the potential to act as novel fetal DNA markers in maternal plasma. Thus, variation in methylation levels at these DMRs regulate normal placentation and placental disorders. Additionally, the methylation at these DMRs might also be susceptible to folic acid supplementation and has the potential to be utilized in clinical diagnosis.

The role of aberrant methylation of trophoblastic stem cell origin in the pathogenesis and diagnosis of placental disorders.

OBJECTIVES: The objective of the study is to investigate the role of methylation levels at promoter regions of placental vascularization genes (VEGF, EGFR, and c-jun) in pathogenesis and diagnosis of placental disorders. METHODS: We analyzed DNA and histone methylation at promoters of VEGF, EGFR, and c-jun via methylation-sensitive high-resolution melting and chromatin immunoprecipitation assay in pregnant women with normal pregnancy in first, second, and third trimesters (n = 30 in each group) and pregnant women with pregnancy complicated with preeclampsia (n = 30) and hydatidiform mole (n = 15). RESULTS: The higher expression of VEGF, EGFR, and c-jun in early pregnancy was observed to be independent of DNA methylation, while it was associated with H3 K9/K27 trimethylations. Also, abnormally higher expression of c-jun in GTDs was associated with lower H3K9me3 level at its promoter. Under preeclampsia conditions, we observed dysregulation of both DNA methylation and H3 trimethylation and subsequent low expression of VEGF, EGFR, and c-jun. Importantly, our promoter methylation data indicated that VEGF may act as novel fetal DNA diagnostic marker for preeclampsia and molar pregnancies in maternal plasma. CONCLUSION: These findings emphasize the importance of dysregulated epigenetic phenomenon behind the pathologies of placental disorders and use of promoter region DNA methylation as an epigenetic marker for these pathological pregnancies. © 2016 John Wiley & Sons, Ltd.

Epigenetic regulation of STAT5A and its role as fetal DNA epigenetic marker during placental development and dysfunction.

INTRODUCTION: Development of normal placenta requires regulated apoptosis of trophoblasts. However, uncontrolled apoptosis has been seen in the pregnancy related complications like hydatidiform mole and pre-eclampsia. STAT5A is a transcription factor with well-known anti-apoptotic role. Thus, we sought to study the role of STAT5A and its epigenetic regulation in placental development and pathologies and its use as fetal DNA epigenetic marker. METHODS: The present study was conducted on pregnant women who were enrolled in five groups, based on the three trimesters in normal pregnancy and two pregnancy related disorder groups: pre-eclampsia and hydatidiform mole. Placental villi samples and maternal blood were obtained from each pregnant woman and were analyzed for promoter region methylation (via methylation sensitive high resolution melting) and histone trimethylations (via chromatin immunoprecipitation) of STAT5A. RESULTS: Our data revealed higher expression of STAT5A in first trimester villi, which decreased with advancing gestation with corresponding increased DNA methylation and H3 trimethylations. Development of choriocarcinoma was associated with DNA methylation associated lower expression of STAT5A. The pattern of promoter methylation of STAT5A in cell free DNA within maternal plasma was observed to be similar to its promoter methylation in placental villi during normal pregnancy, pre-eclampsia and molar complications, which suggested its use as a novel fetal DNA epigenetic marker. DISCUSSION: Our results suggest the regulation of STAT5A via epigenetic mechanisms during normal pregnancy and the association of STAT5A epigenetic dysregulation in pregnancy related complications. Further, hypermethylated STAT5A can be utilized as novel fetal DNA epigenetic marker.

Imbalance between matrix metalloproteinases and their tissue inhibitors in preeclampsia and gestational trophoblastic diseases.

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.

Association of aberrant methylation at promoter regions of tumor suppressor genes with placental pathologies.

AIM: The resemblance between invasive behavior of cancer cells and placental trophoblasts and the role of aberrant epigenetic regulation in cancer development is well known. METHODS: We analyzed the role of promoter region CpG-methylation and H3K9/27me3 of tumor suppressor genes in normal and pathological pregnancies and utilized their CpG-methylation data to search for fetal DNA epigenetic marker in maternal blood. RESULTS: CpG and H3K9/27-methylation associated decreased expression of RASSF1A and APC and increased expression of P16, RB1 and PRKCDBP was observed with advancing normal gestation. Gestational trophoblastic diseases and preeclampsia revealed gene-specific epigenetic deregulation of candidate tumor suppressor genes. Furthermore, APC and PRKCDBP showed the potential to act as fetal DNA epigenetic markers, similar to RASSF1A. CONCLUSION: Deregulation of methylation of tumor suppressor genes contributes to the development of preeclampsia and gestational trophoblastic diseases. APC and PRKCDBP may act as fetal DNA epigenetic markers for prenatal diagnosis.

Gene specific epigenetic regulation of hepatic folate transport system is responsible for perturbed cellular folate status during aging and exogenous modulation.

SCOPE: The present study was designed to identify the molecular mechanism of folate modulation and aging on aberrant liver folate transporter system. METHODS AND RESULTS: An in vivo rat model was used, in which weanling, young and adult rats were given folate deficient diet for 3 and 5 months and after 3 months of folate deficiency, one group received physiological folate repletion (2 mg/kg diet) and another group received over supplemented folate diet (8 mg/kg diet) for another 2 months. In adult group, 3 and 5 months of folate deficiency decreased serum and tissue folate levels with decreased uptake of folate, further associated with decreased expression levels of reduced folate carrier (RFC) and increased expression levels of folate exporter (ABCG2) at both mRNA and protein levels, which in turn regulated by promoter hypermethylation of RFC and promoter hypomethylation of ABCG2 gene. CONCLUSION: Promoter hypermethylation of RFC and promoter hypomethylation of ABCG2 may be attributed to the down regulation of RFC and up regulation of ABCG2 at mRNA and protein levels in conditions of 3 and 5 months of folate deficiency in the adult group.

Increased synthesis of folate transporters regulates folate transport in conditions of ethanol exposure and folate deficiency.

Excessive alcohol consumption and dietary folate inadequacy are the main contributors leading to folate deficiency (FD). The present study was planned to study regulation of folate transport in conditions of FD and ethanol exposure in human embryonic kidney cell line. Also, the reversible nature of effects mediated by ethanol exposure and FD was determined by folate repletion and ethanol removal. For ethanol treatment, HEK293 cells were grown in medium containing 100 mM ethanol, and after treatment, one group of cells was shifted on medium that was free from ethanol. For FD treatment, cells were grown in folate-deficient medium followed by shifting of one group of cells on folate containing medium. FD as well as ethanol exposure resulted in an increase in folate uptake which was due to an increase in expression of folate transporters, i.e., reduced folate carrier, proton-coupled folate transporter, and folate receptor, both at the mRNA and protein level. The effects mediated by ethanol exposure and FD were reversible on removal of treatment. Promoter region methylation of folate transporters remained unaffected after FD and ethanol exposure. As far as transcription rate of folate transporters is concerned, an increase in rate of synthesis was observed in both ethanol exposure and FD conditions. Additionally, mRNA life of folate transporters was observed to be reduced by FD. An increased expression of folate transporters under ethanol exposure and FD conditions can be attributed to enhanced rate of synthesis of folate transporters.

Identification of regulatory mechanisms of intestinal folate transport in condition of folate deficiency.

Folic acid is an essential micronutrient, deficiency of which can lead to disturbance in various metabolic processes of cell. Folate transport across intestine occurs via the involvement of specialized folate transporters viz. proton coupled folate transporter (PCFT) and reduced folate carrier (RFC), which express at the membrane surfaces. The current study was designed to identify the regulatory mechanisms underlying the effects of folate deficiency (FD) on folate transport in human intestinal cell line as well as in rats and to check the reversibility of such effects. Caco-2 cells were grown for five generations in control and FD medium. Following treatment, one subgroup of cells was shifted on folate sufficient medium and grown for three more generations. Similarly, rats were fed an FD diet for 3 and 5 months, and after 3 months of FD treatment, one group of rats were shifted on normal folate-containing diet. Increase in folate transport and expression of folate transporters were observed on FD treatment. However, when cells and rats were shifted to control conditions after treatment, transport and expression of these genes restored to the control level. FD was found to have no impact on promoter methylation of PCFT and RFC; however, messenger RNA stability of transporters was found to be decreased, suggesting some adaptive response. Overall, increased expression of transporters under FD conditions can be attributed to enhanced rate of transcription of folate transporters and also to the increased binding of specificity protein 1 transcription factor to the RFC promoter only.

Reduced SP1-mediated transcriptional activation decreases expression of intestinal folate transporters in response to ethanol exposure.

SCOPE: The study was designed to identify the regulatory mechanisms underlying the effects of ethanol exposure on intestinal folate transport and to investigate the reversibility of such effects. METHODS AND RESULTS: Caco-2 cells were grown in control and ethanol containing medium for 96 h. Thereafter, one subgroup of cells was shifted on ethanol free medium and grown for next 72 h. For in vivo studies, rats were given 1g ethanol/kg body weight/day either for 3 or 5 months and after 3 months of ethanol treatment, one group of rats received no ethanol for 2 months. A significant decrease in folic acid transport as well as expression of folate transporters was observed on ethanol treatment and the effects were reversible upon removal of ethanol. Ethanol exposure had no impact on CpG island methylation of the folate transporters however, an increase in their mRNA half-life was observed that seems to be a homeostatic mechanism. Chromatin immunoprecipitation assay revealed a decrease in binding of SP1 transcription factor to the promoter regions of folate transporters. CONCLUSION: Reduced binding of SP1 to the promoter region of folate transporters may be a part of the regulatory mechanism resulting in decreased expression of folate transporters on ethanol exposure.

Epigenetic mechanisms regulate placental c-myc and hTERT in normal and pathological pregnancies; c-myc as a novel fetal DNA epigenetic marker for pre-eclampsia.

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.

Microsomal Epoxide Hydrolase Polymorphisms and Haplotypes as Determinants of Hepatitis B Virusand Hepatitis C Virus-related Liver Disease in Indian Population.

BACKGROUND: Hepatocellular carcinoma (HCC) is the fifth most common cancer and third leading cause of death worldwide. Main causes of HCC are hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. mEPHX, a xenobiotic metabolizing enzyme, exhibits a dual role of procarcinogen detoxification and activation, hence considered as a cancer risk factor as well as a protective factor. Two known polymorphic forms of mEPHX, exon in exon 3 and 4, are associated with the development of HCC. OBJECTIVE: To determine the association of genotypes and haplotypes of mEPHX with risk of HCC developments separately in HBV- and HCV-infected carriers and patients with hepatitis. METHODS: Polymerase chain reactions (PCR) were carried out using primers to amplify exon 3 (113 Tyr→His variant) and exon 4 (139 His→Arg) polymorphic sites. To distinguish the wild and variant forms, PCR amplification products were digested with restriction endonucleases EcoRV and Rsa1 for exons 3 and 4, respectively. RESULT: Exon 3 genotypes, Y113H and H113H, shared a protective association with HBV-chronic hepatitis infection (P < 0.001 and P< 0.01, respectively) as well as HBV-HCC development (P < 0.001) among HBV-carrier group, while Y113H acts as a risk factor for HCV-chronic hepatitis development (P < 0.001) as well as for HCC development (P < 0.01) with HCV-carrier group as reference. Both H139R and R139R, exon 4 genotypes, acted as a risk factor for HBV/HCV-chronic hepatitis infection and for HBV/HCV-HCC development (P ranges from < 0.05 to < 0.001) with HBV/HCV carriers as reference. 113His-139His and 113His-139Arg haplotypes shared a significant negative and positive association, respectively, with HBV hepatitis and HBV-HCC risk. 113Tyr-139Arg haplotype acted as a risk for HCV-HCC development. CONCLUSION: Polymorphic and haplotypic variant forms of mEPHX exon 3 and 4 variably determine the susceptibility to develop HCC in HBV- and HCV-carrier subjects.