Is there any effect on imprinted genes H19, PEG3, and SNRPN during AOA?

Assisted oocyte activation (AOA) has been proposed as an effective technique to overcome the problem of impaired fertilization after intracytoplasmic sperm injection (ICSI) but the safety of AOA remains a concern. We aimed to investigate if AOA induces imprinting effects on embryos. We used 13 cleavage embryos, nine blastocysts, and eight placentas from 15 patients. The subjects were divided into six groups by tissue type and with or without AOA. The methylation levels of imprinted genes (H19, paternally expressed gene [PEG3] and small nuclear ribonucleoprotein polypeptide N [SNRPN]) were tested by pyrosequencing. We observed different methylation levels among cleavage embryos. The variability was much more remarkable between cleavage embryos than blastocysts and placenta tissues. The methylation levels were especially higher in SNRPN and lower in the H19 gene in AOA embryos than those without AOA. No significant difference was found either among blastocysts or among placenta tissues regardless of AOA. The methylation levels of the three genes in blastocysts were very similar to those in the placenta. Compared to conventional ICSI, AOA changed imprinting methylation rates at H19 and SNRPN in cleavage embryos but not in the blastocyst stage and placenta. We recommend that blastocyst transfer should be considered for patients undergoing AOA during in vitro fertilization.

Orientation of mouse H19 ICR affects imprinted H19 gene expression through promoter methylation-dependent and -independent mechanisms

The mouse Igf2/H19 locus is regulated by genomic imprinting, in which the paternally methylated H19 imprinting control region (ICR) plays a critical role in mono-allelic expression of the genes in the locus. Although the maternal allele-specific insulator activity of the H19 ICR in regulating imprinted Igf2 expression has been well established, the detailed mechanism by which the H19 ICR controls mono-allelic H19 gene expression has not been fully elucidated. In this study, we evaluated the effect of H19 ICR orientation on imprinting regulation in mutant mice in which the H19 ICR sequence was inverted at the endogenous locus. When the inverted-ICR allele was paternally inherited, the methylation level of the H19 promoter was decreased and the H19 gene was derepressed, suggesting that methylation of the H19 promoter is essential for complete repression of H19 gene expression. Unexpectedly, when the inverted allele was maternally inherited, the expression level of the H19 gene was lower than that of the WT allele, even though the H19 promoter remained fully hypomethylated. These observations suggested that the polarity of the H19 ICR is involved in controlling imprinted H19 gene expression on each parental allele, dependent or independent on DNA methylation of the H19 promoter.

P–338 Epigenetic alterations in H19-DMR regulatory region in endometrial tissues of women with endometriosis

Study question Is epigenetic modifications pattern in DMR region of H19 gene different in endometrial tissues of women with endometriosis in compare to normal endometrium? Summary answer The methylation level in DMR region of H19 gene was significantly lower in the endometriosis group. What is known already Endometriosis is characterized by the growth of endometrial-like tissue outside the uterus and has been considered as an epigenetic disease. The lncRNA H19 and insulin-like growth factor–2 (IGF2) genes form a reciprocally imprinted cluster (IGF2/H19). The expression of these two genes is regulated by imprinting control region (ICR). The ICR region is located between these genes and is a differentially methylated region (DMR). The H19 and IGF2 genes are involved in regulating cellular growth and differentiation and might be targeted by MeCP2 (a marker of DNA methylation) for subsequent epigenetic modifications through DMR regulatory region. Study design, size, duration In this case-control study, 12 endometrial samples (eutopic) and 12 endometriotic lesions (ectopic) of women with endometriosis and 12 endometrial control samples were analyzed. Control samples were obtained from women who had no evidence of endometriosis during diagnostic laparoscopy. Control and eutopic endometrial samples were obtained by pipelle. Ectopic samples were obtained during laparoscopy. All women signed the informed consent form and did not receive any hormonal treatments during the last three months. Participants/materials, setting, methods After endometrial tissues collection, gene expression levels of IGF2 and H19 were evaluated using real-time PCR . The occupancy of MeCP2 on two subregions within DMR region of H19 gene was investigated using chromatin immunoprecipitation (ChIP) followed by real-time PCR. One-way ANOVA was used for data analysis. P value less than 0.05 was considered statistically significant. Main results and the role of chance Gene expression profile of H19 and IGF2 was decreased in eutopic and ectopic endometrial lesions of endometriosis group compared with control ones. The decrease in gene expression of H19 in ectopic samples was significant in compared to the control ones while it was nearly significant in compared to the eutopic samples (p = 0.01, p = 0.056, respectively). The ChIP analysis revealed that MeCP2 incorporation in region II (between −3945 and –3818 bp)within DMR region of H19 gene was significantly decreased in eutopic samples compare to control group (p = 0.02) while its decrease was nearly significant in ectopic samples (p = 0.056). However, this DNA methylation profile was not significantly different between eutopic and ectopic endometrial samples in endometriosis in group. Incorporation of MeCP2 in region I (between −2230 and –2103 bp)within DMR region of H19 gene was not significantly different between the eutopic, ectopic and control samples (p > 0.05). (data was presented at 21th Royan International Congress). Limitations, reasons for caution The main limitations of this study is its small sample size. For getting more information, we need to monitor this DNA methylation profile in a large number of women with and without endometriosis. Also, more investigations are required to clarify the further epigenetic modifications in this region. Wider implications of the findings: It seems that reduced expression of IGF2 may be due to hypomethylation of H19-DMR region II while this hypomethylation has no effect on H19 expression in endometriosis. As previously was shown , hypomethylation of H19-DMR causes decrease of IGF2 expression and increase in H19 expression. Trial registration number Not applicable

The long Non-coding RNA Orchestrator of Cancer Axis of Evil Insights into the Multiple Modes of Action of the H19 Gene

Increasing evidence has indicated that the non-coding RNA molecules play central roles in almost all biological processes and many pathological conditions including carcinogenesis. This review focuses on the pathological tumorigenic role of the first discovered long non-coding RNA gene called H19 and its pivotal contribution to the cancer axis of evil. H19 RNA utilizes a variety of mechanisms to perform its pathological function. Some key unanswered questions are presented by the end. Understanding the H19 RNA mechanisms of action will shed light into the class of long non-coding RNA which contains thousands of members mostly with unknown function and will help in delineating the pathological role played by at least some of them.

Epigenetic characterization of the H19/IGF2 locus in calf clones placenta

ABSTRACT: Somatic Cell Nuclear Transfer (SCNT-Cloning) is a promising technique in many areas and is based on genetically identical individuals. However, its efficiency is low. Studies suggest that the leading cause is inadequate epigenetic reprogramming. This study aimed to characterize the methylation pattern of the exon 10 regions of the IGF2 gene and the Imprinting Control Region (ICR) of the H19 gene in the placenta of cloned calves. For this study, female and male cloned calves presenting different phenotypes were used. Genomic DNA from these animals’ placenta was isolated, then treated with sodium bisulfite and amplified to the ICR/H19 and IGF2 loci. PCR products were cloned into competent bacteria and finally sequenced. A significant difference was found between controls and clones with healthy phenotypes for the ICR/H19 region. In this region, controls showed a hemimethylated pattern, as predicted in the literature due to this region has an imprinted control, while clones were showed less methylated. For the IGF2, no significant differences were found between controls and clones. These results suggest that different genomic regions in the genome may be independently reprogrammed and that failures in reprogramming the DNA methylation patterns of imprinted genes may be one of the causes of the low efficiency of SCNT.

Long Non-Coding RNA H19 Expression and Functional Polymorphism rs217727 Are Linked to Increased Ischemic Stroke Risk

Background: Efforts to identify potential biomarkers for the diagnosis of ischemic stroke (IS) are valuable. The H19 gene plays a functional role in increasing the prevalence of IS risk factors. We evaluated the correlation between H19 rs217727 polymorphism and the expression level of H19 lncRNA with susceptibility to IS among the Iranian population.Methods: Blood samples were collected from IS patients (n = 114) and controls (n = 114). We concentrated on the expression pattern of H19 at different time points (i.e., 0-24, 24-48, and 48-72 hours after stroke). The tetra-primer ARMS-PCR method was applied for DNA genotyping. We used the quantitative real-time PCR to evaluate H19 expression levels. Results: The rs217727polymorphism of H19 was related with IS susceptibility in the co-dominant (OR=2.92, 95% CI=0.91-10.92, P=0.04) and recessive models (OR=2.80, 95% CI=0.96-8.15, P=0.04). H19 expression was significantly upregulated in IS and remained high for 72 hours after stroke. ROC curves showed that H19 expression within the first 24 hours from stroke onset might serve as a biomarker for the early diagnosis of IS with 79.49% sensitivity and 80.00% specificity. H19 expression in SVO and LAA patients were 3.74 and 3.34 times higher than the UD subtype, respectively [OR=3.74 95% CL (1.14-12.27) P=0.030 and OR=3.34 95% CL (1.13-9.85) P=0.029].Conclusion: The rs217727 polymorphism of the H19 is correlated with IS susceptibility, and H19 expression levels were higher in SVO and LAA patients. The upregulation of H19 may be considered as a diagnostic biomarker in IS among the Iranian population.