Imprinting Of Insulin-like Growth Factor Research Articles

Effects of methylation and imprinting expression of Insulin-like growth factor 2 gene in gastric cancer.

Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) is a common malignant tumor associated with EBV infection. Insulin-like growth factor 2 (IGF2) is an imprinted gene and a key protein that regulates growth, especially during normal fetal development. Loss of imprinting (LOI), is a common epigenetic anomaly in a variety of human cancers. However, the promoter methylation, imprinting status and function of IGF2 gene in GC are unclear. To explore the role of IGF2 in the occurrence and development of gastric cancer. The biological function of IGF2 in gastric cancer was investigated by Transwell, wound healing, CCK-8 and flow cytometry assays. IGF2 imprinting status and gene promoter methylation in gastric cancer tissues were detected by PCR-RFLP and BGS. The results showed that the expression of IGF2 was higher in GC tissues than adjacent tissues. IGF2 gene promoter methylation and LOI were significantly higher in EBVaGC tissues than in EBV-negative gastric cancer (EBVnGC) tissues. The high expression of IGF2 in gastric cancer can promote the migration and proliferation of gastric cancer cells. Our data suggest that IGF2 is involved in the occurrence and development of gastric cancer. Targeting IGF2 may be a potential therapeutic target for gastric cancer.

Effects of IGF2 hP4 promoter targeted methylation on imprinting status of renal cell carcinoma

Objective To investigate the imprinting status of insulin-like growth factor 2 (IGF2) in renal cell carcinoma ACHN cell line, as well as the effect of methylated drug TCF1001 on methylation status of IGF2 hP4 promoter. Furthermore, the relationship between hP4 promoter methylation and imprinting status change of IGF2 gene was also examined. Methods The heterozygote status of IGF2 gene was detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The imprinting status of IGF2 was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and PCR-RFLP. ACHN cells were then treated with either the methylated drug TCF1001 or control drugs (CT010 or PBS). Bisulfite sequencing was used to study the methylation status of IGF2 hP4 promoter region. Meanwhile, PCR-RFLP was for analyzing the variation of IGF2 imprinting status. Results It was found that IGF2 of ACHN cells was in the status of loss of imprinting (LOI). TCF1001 could effectively induce methylation of IGF2 hP4 promoter. Furthermore, the LOI of IGF2 could be reversed when IGF2 hP4 promoter was partially methylated. Conclusions IGF2 hP4 promoter can be targeted methylated by TCF1001. IGF2 hP4 promoter targeted methylation can partially reverse the LOI of IGF2, which provides the theoretical basis of renal cell carcinoma genetic therapy. Key words: Kidney Neoplasms; Insulin-Like Growth Factor II; Genomic Imprinting; Methylation

Rs10732516 polymorphism at the IGF2/H19 locus associates with genotype-specific effects on placental DNA methylation and birth weight of newborns conceived by assisted reproductive technology

BackgroundAssisted reproductive technology (ART) has been associated with low birth weight of fresh embryo transfer (FRESH) derived and increased birth weight of frozen embryo transfer (FET)-derived newborns. Owing to that, we focused on imprinted insulin-like growth factor 2 (IGF2)/H19 locus known to be important for normal growth. This locus is regulated by H19 imprinting control region (ICR) with seven binding sites for the methylation-sensitive zinc finger regulatory protein (CTCF). A polymorphism rs10732516 G/A in the sixth binding site for CTCF, associates with a genotype-specific trend to the DNA methylation. Due to this association, 62 couples with singleton pregnancies derived from FRESH (44 IVF/18 ICSI), 24 couples from FET (15 IVF/9 ICSI), and 157 couples with spontaneously conceived pregnancies as controls were recruited in Finland and Estonia for genotype-specific examination. DNA methylation levels at the H19 ICR, H19 DMR, and long interspersed nuclear elements in placental tissue were explored by MassARRAY EpiTYPER (n = 122). Allele-specific changes in the methylation level of H19 ICR in placental tissue (n = 26) and white blood cells (WBC, n = 8) were examined by bisulfite sequencing. Newborns’ (n = 243) anthropometrics was analyzed by using international growth standards.ResultsA consistent trend of genotype-specific decreased methylation level was observed in paternal allele of rs10732516 paternal A/maternal G genotype, but not in paternal G/maternal A genotype, at H19 ICR in ART placentas. This hypomethylation was not detected in WBCs. Also genotype-specific differences in FRESH-derived newborns’ birth weight and head circumference were observed (P = 0.04, P = 0.004, respectively): FRESH-derived newborns with G/G genotype were heavier (P = 0.04) and had larger head circumference (P = 0.002) compared to newborns with A/A genotype. Also, the placental weight and birth weight of controls, FRESH- and FET-derived newborns differed significantly in rs10732516 A/A genotype (P = 0.024, P = 0.006, respectively): the placentas and newborns of FET-derived pregnancies were heavier compared to FRESH-derived pregnancies (P = 0.02, P = 0.004, respectively).ConclusionsThe observed DNA methylation changes together with the phenotypic findings suggest that rs10732516 polymorphism associates with the effects of ART in a parent-of-origin manner. Therefore, this polymorphism should be considered when the effects of environmental factors on embryonic development are studied.

The long non-coding RNA H19 suppresses carcinogenesis and chemoresistance in hepatocellular carcinoma

The long non-coding RNA (lncRNA) H19 represents a maternally expressed and epigenetically regulated imprinted gene product and is discussed to have either tumor-promoting or tumor-suppressive actions. Recently, H19 was shown to be regulated under inflammatory conditions. Therefore, aim of this study was to determine the function of H19 in hepatocellular carcinoma (HCC), an inflammation-associated type of tumor. In four different human HCC patient cohorts H19 was distinctly downregulated in tumor tissue compared to normal or non-tumorous adjacent tissue. We therefore determined the action of H19 in three different human hepatoma cell lines (HepG2, Plc/Prf5, and Huh7). Clonogenicity and proliferation assays showed that H19 overexpression could suppress tumor cell survival and proliferation after treatment with either sorafenib or doxorubicin, suggesting chemosensitizing actions of H19. Since HCC displays a highly chemoresistant tumor entity, cell lines resistant to doxorubicin or sorafenib were established. In all six chemoresistant cell lines H19 expression was significantly downregulated. The promoter methylation of the H19 gene was significantly different in chemoresistant cell lines compared to their sensitive counterparts. Chemoresistant cells were sensitized after H19 overexpression by either increasing the cytotoxic action of doxorubicin or decreasing cell proliferation upon sorafenib treatment. An H19 knockout mouse model (H19Δ3) showed increased tumor development and tumor cell proliferation after treatment with the carcinogen diethylnitrosamine (DEN) independent of the reciprocally imprinted insulin-like growth factor 2 (IGF2). In conclusion, H19 suppresses hepatocarcinogenesis, hepatoma cell growth, and HCC chemoresistance. Thus, mimicking H19 action might be a potential target to overcome chemoresistance in future HCC therapy.

Gene therapy for colorectal cancer by adenovirus-mediated siRNA targeting CD147 based on loss of the IGF2 imprinting system.

Colorectal cancer (CRC) is one of the most common malignant tumors worldwide. Loss of imprinting (LOI) of the insulin-like growth factor 2 (IGF2) gene is an epigenetic abnormality phenomenon in CRC. Recently observed association of CRC with cluster of differentiation 147 (CD147) could provide a novel approach for gene therapy. In the present study, we investigated the feasibility of using adenovirus‑mediated siRNA targeting CD147 based on the IGF2 LOI system for targeted gene therapy of CRC. A novel adenovirus-mediated siRNA targeting CD147, rAd-H19-CD147mirsh, which was driven by the IGF2 imprinting system, was constructed. The results showed that the EGFP expression was detected only in the IGF2 LOI cell lines (HT-29 and HCT-8), but that no EGFP was produced in cell lines with maintenance of imprinting (MOI) (HCT116). Moreover, rAd-H19-CD147mirsh significantly inhibited the expression of CD147, decreased cell viability and invasive ability, and increased sensitivity to chemotherapeutic drugs only in the LOI cell lines in vitro. Furthermore, mice bearing HT-29 xenografted tumors, which received intratumoral administration of the rAd-H19-CD147mirsh, showed significantly reduced tumor growth and enhanced survival. We conclude that recombinant adenovirus-mediated siRNA targeting CD147 based on the IGF2 LOI system inhibited the growth of the LOI cells in vitro and in vivo, which would provide a novel approach for targeted CRC gene therapy.

Gene therapy for human colorectal cancer cell lines with recombinant adenovirus 5 based on loss of the insulin-like growth factor 2 imprinting.

The recombinant oncolytic adenovirus is a novel anticancer agent to replicate selectively in colon cancer cell lines. Loss of imprinting (LOI) of insulin-like growth factor 2 (IGF2) gene is an epigenetic abnormality phenomenon. We utilized the IGF2 LOI in gene therapy for the malignant tumor cell lines. We investigated the tumoricidal effects of IGF2 LOI on four cell lines by oncolytic adenovirus, and constructed novel adenovirus vectors Ad312-E1A and Ad312-EGFP. The expression of E1A was monitored by real-time PCR and western blot analysis. The viability and apoptosis of colorectal cells infected with Ad312-E1A were tested by CCK-8 and flow cytometry. In addition, we established a colorectal cancer model in nude mice. The results showed that HCT-8 and HT-29 with IGF2 LOI were infected with Ad312-EGFP and then produced the EGFP. Nevertheless, SW480 and GES-1, which were IGF2 MOI, did not produce the EGFP. The Ad312-E1A obviously reduced the cell viability and induced apoptosis in HCT-8 and HT-29 in vitro, and successfully suppressed tumor growth in HT-29 xenografts in nude mice. In summary, the conditionally replicative adenovirus with loss of IGF2 imprinting system has a positive effect on gene therapy.

Association of imprinting status of IGF2 with the pathophysiology of intrauterine growth retardation in rats

Insulin like growth factor 2 (IGF2) is a crucial imprinting gene which prompting placenta development and fetal growth. It demonstrates parent-of-origin-specific allelic expression that is epigenetically regulated by differential methylation of IGF2/H19 DMR. We aim to explore if abnormal IGF2 imprinting status that regulated by altered DNA methylation of IGF2/H19 DMR affects IGF2 expression leading to the occurrence of IUGR. First we established an IUGR model of rat at two different stages(19-d gestational age and 1-day-old) by maternal nutrition restriction, and the rat tissues in the control group and the IUGR group were collected which were divided into four subgroups(N=6~8, each subgroup): Subgroup 1 and Subgroup 2 was the placenta and liver tissue of fetuses with 19-d gestational age, respectively, Subgroup 3 and Subgroup 4 was the liver tissue and skeletal muscle tissue in the posterior limbs of 1-day-old rats, respectively. Then the mRNA and protein levels of IGF2 were tested in the four subgroups of the two groups. In each subgroup, pyrosequencing was used to test the DNA methylation level of imprinting control region IGF2/H19 DMR. We observed that the expression levels of IGF2 in the Subgroup1, Subgroup 2, Subgroup 3 were decreased significantly compared with the control group, but there were no obvious differences in the Subgroup 4 of the two groups. The pyrosequencing showed no significant differences in methylation levels of the CpG dinucleotides in IGF2/H19 DMR between the two groups. What’s more, the methylation ratio didn't change versus age, and maintained the same from fetus till adult, and it also stayed the same among different tissue types except for placenta which had a lower methylation fraction than the other tissues. We concluded that the expression of IGF2 was down-regulated in the IUGR rats, which could play an important role in the pathogenesis of IUGR. However, the down regulation was not always the result of the aberrant imprinting status of the imprinting control region H19 DMR, as there wasn't a clear association between the two. IGF2 could be down regulated through other pathways, such as histone modification, or altered imprinting of another control region, to mediate the development of IUGR.

Altered Methylation of IGF2 Locus 20 Years after Preterm Birth at Very Low Birth Weight

IntroductionPeople born preterm at very low birth weight (VLBW, ≤1500g) have higher rates of risk factors for adult-onset diseases, including cardiovascular diseases and type 2 diabetes. These risks may be mediated through epigenetic modification of genes that are critical to normal growth and development.MethodsWe measured the methylation level of an imprinted insulin-like-growth-factor 2 (IGF2) locus (IGF2/H19) in young adults born preterm at VLBW and in their peers born at term. We studied 158 VLBW and 161 control subjects aged 18 to 27 years from the Helsinki Study of Very Low Birth Weight Adults. Methylation fraction at two IGF2 differentially methylated regions (DMRs) – IGF2 antisense transcript (IGF2AS, also known as IGF2 DMR0) and last exon of IGF2 (IGF2_05, also known as IGF2 DMR2) – were measured with Sequenom Epityper. We used linear regression and adjustment for covariates to compare methylation fractions at these DMRs between VLBW and control subjects.ResultsAt one IGF2AS CpG site, methylation was significantly lower in VLBW than in control subjects, mean difference −0.017 (95% CI; −0.028, −0.005), P = 0.004. Methylation at IGF2_05 was not different between the groups.ConclusionsMethylation of IGF2AS is altered 20 years after preterm birth at VLBW. Altered methylation may be a mechanism of later increased disease risk but more data are needed to indicate causality.

Abstract 5354: Altered DNA methylation at imprinted insulin-like growth factor-2 (IGF2) resulting from heavy metal exposure in vitro and in vivo in infants from the newborn epigenetic study (NEST).

Abstract Early life exposure to heavy metals, including lead, has adverse consequences on health and development but the mechanism(s) underlying these effects are unclear. Epigenetic deregulation resulting from lead exposure may underlie these effects, and imprinted genes may be particularly vulnerable since imprinting is established and maintained in large part through DNA methylation. Imprinted genes are powerful regulators of growth and energy utilization. The best studied imprinted genes are insulin-like growth factor 2 (IGF2) and H19 which are regulated by at least two differentially methylated regions (DMRs). Altered methylation at the IGF2/H19 DMRs has been associated with obesity, neurodevelopmental disorders, and cancer. Data from the Newborn Epigenetics STudy (NEST), which is focused on the influence of early life environmental exposures on children's health, has indicated that epigenetic alterations resulting from environmental exposures can be sex-specific and exacerbated in African Americans as compared to whites. We sought to determine if in vitro exposure to lead alters DNA methylation at two regulatory DMRs important to imprinting and expression of IGF2 and to assess how these results compared to the effects of lead exposure in the NEST cohort. Immortalized normal human embryonic kidney cells (HEK-293) were exposed to lead acetate (0-25 μg/dL) for up to 72 hours followed by purification of genomic DNA. Bisulfite modification of HEK-293 and NEST cohort DNA specimens was followed by pyrosequencing to quantify DNA methylation. The North Carolina State Laboratory for Public Health performed the blood lead level testing as part of a routine check-up for children between the ages of 1-3 years old. The NEST cohort participants were born between 2005-2008 at obstetrics-care facilities in Durham County, North Carolina. For this study we used a sub-population of participants whose mother lived at the same address since conception in order to more accurately determine early and consistent environmental lead exposure. Analysis of variance (ANOVA) testing was done on the in vitro data to examine the difference between mean values of the lead treatment conditions. For the NEST samples we used generalized linear models in a sample of n=182 participants to examine aberrant methylation at the IGF2 DMRs in children exposed to environmental lead from conception to ages 1-3 years. In our in vitro model of early environmental lead exposure we saw a 3-5% decrease in IGF2 DMR methylation with increasing lead acetate concentration. Similar to the hypomethylation seen in the in vitro studies, within the NEST cohort we observed a 4% decrease in methylation at the H19 DMR in female children with blood lead levels between 2-4 μg/dL and an 8% decrease in methylation for female infants with blood lead levels equal to or higher than 5 μg/dL of lead. Citation Format: Monica D. Nye, Adriana Vidal, Cathrine Hoyo, Susan K. Murphy. Altered DNA methylation at imprinted insulin-like growth factor-2 (IGF2) resulting from heavy metal exposure in vitro and in vivo in infants from the newborn epigenetic study (NEST). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5354. doi:10.1158/1538-7445.AM2013-5354

Paternal obesity is associated with IGF2hypomethylation in newborns: results from a Newborn Epigenetics Study (NEST) cohort

BackgroundData from epidemiological and animal model studies suggest that nutrition during pregnancy may affect the health status of subsequent generations. These transgenerational effects are now being explained by disruptions at the level of the epigenetic machinery. Besides in vitro environmental exposures, the possible impact on the reprogramming of methylation profiles at imprinted genes at a much earlier time point, such as during spermatogenesis or oogenesis, has not previously been considered. In this study, our aim was to determine associations between preconceptional obesity and DNA methylation profiles in the offspring, particularly at the differentially methylated regions (DMRs) of the imprinted Insulin-like Growth Factor 2 (IGF2) gene.MethodsWe examined DNA from umbilical cord blood leukocytes from 79 newborns, born between July 2005 and November 2006 at Duke University Hospital, Durham, NC. Their mothers participated in the Newborn Epigenetics Study (NEST) during pregnancy. Parental characteristics were obtained via standardized questionnaires and medical records. DNA methylation patterns at two DMRs were analyzed by bisulfite pyrosequencing; one DMR upstream of IGF2 (IGF2 DMR), and one DMR upstream of the neighboring H19 gene (H19 DMR). Multiple regression models were used to determine potential associations between the offspring's DNA methylation patterns and parental obesity before conception. Obesity was defined as body mass index (BMI) ≥30 kg/m2.ResultsHypomethylation at the IGF2 DMR was associated with paternal obesity. Even after adjusting for several maternal and newborn characteristics, we observed a persistent inverse association between DNA methylation in the offspring and paternal obesity (β-coefficient was -5.28, P = 0.003). At the H19 DMR, no significant associations were detected between methylation patterns and paternal obesity. Our data suggest an increase in DNA methylation at the IGF2 and H19 DMRs among newborns from obese mothers, but a larger study is warranted to further explore the potential effects of maternal obesity or lifestyle on the offspring's epigenome.ConclusionsWhile our small sample size is limited, our data indicate a preconceptional impact of paternal obesity on the reprogramming of imprint marks during spermatogenesis. Given the biological importance of imprinting fidelity, our study provides evidence for transgenerational effects of paternal obesity that may influence the offspring's future health status.

Gene therapy for colorectal cancer by an oncolytic adenovirus that targets loss of the insulin-like growth factor 2 imprinting system

BackgroundColorectal cancer is one of the most common malignant tumors worldwide. Loss of imprinting (LOI) of the insulin-like growth factor 2 (IGF2) gene is an epigenetic abnormality observed in human colorectal neoplasms. Our aim was to investigate the feasibility of using the IGF2 imprinting system for targeted gene therapy of colorectal cancer.ResultsWe constructed a novel oncolytic adenovirus, Ad315-E1A, and a replication-deficient recombinant adenovirus, Ad315-EGFP, driven by the IGF2 imprinting system by inserting the H19 promoter, CCCTC binding factor, enhancer, human adenovirus early region 1A (E1A) and enhanced green fluorescent protein (EGFP) reporter gene into a pDC-315 shuttle plasmid. Cell lines with IGF2 LOI (HCT-8 and HT-29), which were infected with Ad315-EGFP, produced EGFP. However, no EGFP was produced in cell lines with maintenance of imprinting (HCT116 and GES-1). We found that Ad315-E1A significantly decreased cell viability and induced apoptosis only in LOI cell lines in vitro. In addition, mice bearing HCT-8-xenografted tumors, which received intratumoral administration of the oncolytic adenovirus, showed significantly reduced tumor growth and enhanced survival.ConclusionsOur recombinant oncolytic virus targeting the IGF2 LOI system inhibits LOI cell growth in vitro and in vivo, and provides a novel approach for targeted gene therapy.

Abstract B35: Altered DNA methylation at imprinted insulin-like growth factor-2 (IGF2) resulting from heavy metal exposure in vitro and in vivo in infants from the Newborn Epigenetic STudy (NEST).

Abstract Background: Early life exposure to heavy metals, including lead, has adverse consequences on health and development but the mechanism(s) underlying these effects are unclear. Epigenetic deregulation resulting from lead exposure may underlie these effects, and imprinted genes may be particularly vulnerable since imprinting is established and maintained in large part through DNA methylation. The best studied imprinted regulatory region is the insulin-like growth factor 2-H19 (IGF2) domain that is regulated by at least two differentially methylated regions (DMRs). Altered methylation at the IGF2 DMRs has been associated with obesity, neurological development disorders, and cancer. Data from the Newborn Epigenetics STudy (NEST), which is focused on the influence of early life environmental exposures on children's health. This study has indicated that epigenetic alterations resulting from these environmental exposures are often sex-specific and exacerbated in African Americans as compared to whites. Objective: We sought to determine if in vitro exposure to lead alters DNA methylation at two regulatory DMRs is important to imprinting and expression of IGF2 and to assess how these results compared to the effects of lead exposure in the NEST cohort. Methods: Immortalized normal human embryonic kidney cells (HEK293) were exposed to lead acetate (0-25 ug/dL) for 48-72 hours followed by purification of genomic DNA. Bisulfite modification of HEK293 and NEST cohort DNA specimens was followed by pyrosequencing to quantify DNA methylation. The North Carolina State Laboratory for Public Health performed the blood lead level testing as part of a routine check-up for children between the ages of 2-3 years old. The NEST cohort participants were born between 2005-2008 at obstetrics-care facilities in Durham County, North Carolina. For this study we used a sub-population of participants whose mother lived at the same address since conception in order to more accurately determine early and consistent environmental lead exposure. Analysis of variance (ANOVA) tests were done on the in vitro data to examine the difference between mean values of the lead treatment conditions. For the NEST samples we used generalized linear models in a sample of n=182 participants to examine aberrant methylation at the IGF2 DMRs, in children exposed to environmental lead from conception to age 2-3 years old. Results: In our in vitro model of early environmental lead exposure we saw a 3-5% decrease (p= 0.006) in IGF2 DMRs methylation profiles with increasing lead acetate concentration. Similar to the in vitro studies, in the NEST cohort we observed 4% decrease (p <0.0001) in methylation at the H19 DMRs of IGF2/H19 gene in female children with blood lead levels between 2-4 ug/dL and an 8% decrease (p<0.0001) in methylation for female infants with blood lead levels equal to or higher than 5 ug/dL of lead. Conclusions: Lead exposure was associated with aberrant methylation at the IGF2/H19 DMR in vitro and in a cohort study of young children exposed to lead in their home environment pre and post-natal. The implications of these findings in vitro and in vivo suggest a link between lead exposure and altered methylation at the IGF2/H19 DMRs. Larger studies are required. Citation Format: Monica D. Nye, Adriana C. Vidal, Marilie D. Gammon, Cathrine Hoyo. Altered DNA methylation at imprinted insulin-like growth factor-2 (IGF2) resulting from heavy metal exposure in vitro and in vivo in infants from the Newborn Epigenetic STudy (NEST). [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr B35.

Loss of imprinting of IGF2 correlates with hypomethylation of the H19 differentially methylated region in the tumor tissue of colorectal cancer patients

Expression of the imprinted genes insulin-like growth factor 2 (IGF2) and H19 depends on the methylation pattern of their differentially methylated region (DMR) located on chromosome 11p15. In the present study, we examined the imprinting status of the IGF2 gene in 120 human colorectal cancer (CRC) patients and 150 normal controls. In addition, we analyzed the DNA methylation of the sixth CTCF-binding site in the DMR of IGF2/H19 in 81 CRC patients using bisulfate sequencing. Of a total of 81 informative (heterozygous) samples, 51 samples showed bialleic IGF2 expression in tumor samples; however, only 15 of 69 informative samples showed loss of imprinting (LOI) of IGF2 in normal controls. Statistically significant differences in the methylation status between the retention of imprinting (ROI) and LOI groups (66.1±14.9 vs. 16.7±9.2, p=0.008) were observed. The results of the present study suggest that LOI of IGF2 is important in the carcinogenesis of CRC. Hypomethylation of the sixth CTCF-binding site in the DMR of IGF2/H19 is linked to LOI and the common IGF2-H19 enhancer competition model for IGF2 imprinting does not apply to human CRC.

Association of cord blood methylation fractions at imprinted insulin-like growth factor 2 (IGF2), plasma IGF2, and birth weight

PurposeAltered methylation at Insulin-like Growth Factor 2 (IGF2) regulatory regions has previously been associated with obesity, and several malignancies including colon, esophageal, and prostate adenocarcinomas, presumably via changes in expression and/or loss of imprinting, but the functional significance of these DNA methylation marks have not been demonstrated in humans. We examined associations among DNA methylation at IGF2 differentially methylated regions (DMRs), circulating IGF2 protein concentrations in umbilical cord blood (UCB) and birth weight in newborns.MethodsQuestionnaire data were obtained from 300 pregnant women recruited between 2005 and 2009. UCB DNA methylation was measured by bisulfite pyrosequencing. UCB plasma concentrations of soluble IGF2 were measured by ELISA assays. Generalized linear regression models were used to examine the relationship between DMR methylation and IGF2 levels.ResultsLower IGF2 DMR methylation was associated with elevated plasma IGF2 protein concentrations (β = −9.87, p < 0.01); an association that was stronger in infants born to obese women (pre-pregnancy BMI > 30 kg/m2, β = −20.21, p < 0.0001). Elevated IGF2 concentrations were associated with higher birth weight (p < 0.0001) after adjusting for maternal race/ethnicity, pre-pregnancy BMI, cigarette smoking, gestational diabetes, and infant sex. These patterns of association were not apparent at the H19 DMR.ConclusionOur data suggest that variation in IGF2 DMR methylation is an important mechanism by which circulating IGF2 concentrations, a putative risk factor for obesity and cancers of the colon, esophagus, and prostate, are modulated; associations that may depend on pre-pregnancy obesity.

The effects of depression and use of antidepressive medicines during pregnancy on the methylation status of the IGF2 imprinted control regions in the offspring

In utero exposures to environmental factors may result in persistent epigenetic modifications affecting normal development and susceptibility to chronic diseases in later life. We explored the relationship between exposure of the growing fetus to maternal depression or antidepressants and DNA methylation at two differentially methylated regions (DMRs) of the imprinted Insulin-like Growth Factor 2 (IGF2) gene. Aberrant DNA methylation at the IGF2 and neighboring H19 DMRs has been associated with deregulated IGF2 expression, childhood cancers and several chronic diseases during adulthood. Our study population is comprised of pregnant mothers and their newborns (n = 436), as part of the Newborn Epigenetics Study (NEST). A standardized questionnaire was completed and medical record data were abstracted to ascertain maternal depression and antidepressive drug use. DMR methylation levels in umbilical cord blood leukocytes were quantified using pyrosequencing. From the 436 newborns, laboratory data were obtained for 356 individuals at the IGF2 DMRs, and for 411 individuals at the H19 DMRs; about half of each group was African American or Caucasian. While overall no association between depression and methylation profiles was found, we observed a significant hypermethylation of the H19 DMRs in newborns of African American (n = 177) but not Caucasian (n = 168) mothers who reported the use of antidepressive drugs during pregnancy (β = +6.89, p = 0.01). Of note, our data reveal a race-independent association between smoking during pregnancy and methylation at the IGF2 DMR (+3.05%, p = 0.01). In conclusion, our findings suggest a race-dependent response related to maternal use of antidepressants at one of the IGF2 DMRs in the offspring.

H19/miR-675 non-coding RNA expression differentiates among cancers of the human endometrium.

H19 is a maternally expressed non-coding RNA located at chromosome 11p15.5 near the reciprocally imprinted insulin-like growth factor 2 (IGF2) gene. Though the function of H19 is unknown, it is transcribed during embryonic development after which transcription is absent in all but a few tissues including cardiac muscle, breast, ovary, uterus, and placenta. Linking H19, <em>miR-675</em> and RB1 expression with serous tumors of the endometrium suggests that RB1 suppression may be a differentiating event in serous tumorigenesis.

Abstract 2754: Maternal use of antidepressants in pregnancy is associated with hypermethylation at the IGF2 imprinted control regions in the offspring in a race-dependent fashion

Abstract In utero exposures to environmental factors may result in persistent epigenetic modifications affecting normal development and susceptibility to chronic diseases in later life. We determined whether exposure of the growing fetus to maternal depression or antidepressants is associated with aberrant DNA methylation at two differentially methylated regions (DMRs) of the imprinted Insulin-like Growth Factor 2 (IGF2) gene, namely the DMRs upstream of the imprinted IGF2 promoters (IGF2 DMR) and the DMRs upstream of H19 (H19 DMR). Aberrant DNA methylation at these DMRs has been associated with deregulated IGF2 expression, childhood cancers and several chronic diseases during adulthood. Our study population comprises pregnant mothers and their newborns (n=436), recruited between 2005 and 2008 in Durham, NC. A standardized questionnaire was completed and medical record data were abstracted to ascertain prenatal and periconceptional depression and antidepressive drug use. DMR methylation percentages in umbilical cord blood leukocytes were quantified using pyrosequencing. While an association between depression and methylation profiles was not found, we discovered significant hypermethylation of the H19 DMRs in newborns of African American -but not Caucasian- mothers who were taking antidepressive drugs during pregnancy (β=+6.89, p=0.01). Our findings suggest that changes in the IGF2/H19-domain methylation in response to maternal exposure to antidepressants are race-dependent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2754. doi:10.1158/1538-7445.AM2011-2754

Insulin‐like growth factor‐2 (IGF2) loss of imprinting marks a field defect within human prostates containing cancer

Loss of imprinting (LOI) is an epigenetic alteration involving loss of parental origin-specific expression at normally imprinted genes. A LOI for IGF2, a paracrine growth factor, has been implicated in the development of prostate and other cancers. In the current study, we define IGF2 LOI in histologically normal prostate tissues in relationship to tumor foci and gene expression. Microdissected tumor associated (TA) adjacent (2 mm) and distant (10 mm) tissues surrounding tumor foci were generated. IGF2 imprinting in informative prostate tissue sets was quantitated using a fluorescent primer extension assay and expression analyzed utilizing quantitative PCR. DNA methylation analyses were performed using quantitative pyrosequencing. A marked IGF2 LOI was found in adjacent TA tissues (39 ± 3.1%) and did not significantly decrease in tissues distant (38 ± 5.3%) from tumor foci (45 ± 2.9%; P = 0.21). IGF2 imprinting correlated with IGF2 expression in TA tissues, but not within the tumor foci. Hypomethylation of the IGF2 DMR0 region correlated with decreased IGF2 expression in tumors (P < 0.01). The expression of IGF2 and its adjacent imprinted gene H19 were increased in adjacent and distant tissues compared to tumors (P < 0.05) indicating the importance of factors other than LOI in driving IGF2 expression. LOI of IGF2 occurs not only adjacent to prostate tumor foci, but is widely prevalent even in distant areas within the peripheral zone. These data provide evidence for a widespread epigenetic field defect in histologically normal tissues that might be employed to identify prostate cancer in patients.

Imprinting and Promoter Usage of Insulin‐Like Growth Factor II in Twin Discordant Placenta

Case reports from infant twins suggest that abnormal genomic imprinting may be one of the important causes of twin discordance, but it is unknown whether abnormal genomic imprinting occurs in the placenta. Therefore, we sought to determine the relationship between the imprinting of insulin-like growth factor II (IGF-II) in placenta and twin discordance. We analyzed the imprinting and promoter usage of IGF-II in placenta of normal twins (T0 group), weight discordance (T1 group), and phenotype discordance (T2 group). We found the incidence of loss of imprinting (LOI) for IGF-II was higher in the T2 group than that in the T0 and T1 groups, while there was no difference between T0 and T1 groups. The transcripts of promoter 3 were lower in the T2 group than in the T0 and T1 groups, and lower in the twin placenta with LOI than in those with normal imprinting. Our findings indicate that the promoter 3 specific LOI of the IGF-II gene may be closely related with phenotype discordance, not weight discordance.

Transient in vitro epigenetic reprogramming of skin fibroblasts into multipotent cells

Multipotent stem cells have the potential to establish a new field of promising regenerative medicine to treat tissue damage, genetic disorders, and degenerative diseases. However, limited resource of stem cells has turned to be an evitable obstacle in clinical applications. We utilized a simple in vitro epigenetic reprogramming approach to convert skin fibroblasts into multipotent cells. After transient reprogramming, stem cell markers, including Oct4 and Nanog, became activated in the treated cells. The reprogrammed cells were multipotent as demonstrated by their ability to differentiate into a variety of cells and to form teratomas. Genomic imprinting of insulin-like growth factor II ( Igf2) and H19 was not affected by this short period of cell reprogramming. This study may provide an alternative strategy to efficiently generate patient-specific stem cells for basic and clinical research, solving major hurdles of virally-induced pluripotent stem (iPS) cells that entail the potential risks of mutation, gene instability, and malignancy.