Gene expression and epigenetic aberrations in F1-placentas fathered by obese males.

Gene expression and/or epigenetic deregulation may have consequences for sperm and blastocysts, as well as for the placenta, together potentially contributing to problems observed in offspring. We previously demonstrated specific perturbations of fertilization, blastocyst formation, implantation, as well as aberrant glucose metabolism and adiposity in offspring using a mouse model of paternal obesity. The current investigation analyzed gene expression and methylation of specific CpG residues in F1 placentas of pregnancies fathered by obese and normal-weight male mice, using real-time PCR and bisulfite pyrosequencing. Our aim was to determine if paternal obesity deregulated placental gene expression and DNA methylation when compared to normal-weight males. Gene methylation of sperm DNA was analyzed and compared to placentas to address epigenetic transmission. Of the 10 paternally expressed genes (Pegs), 11 genes important for development and transport of nutrients, and the long-terminal repeat Intracisternal A particle (IAP) elements, derived from a member of the class II endogenous retroviral gene family, we observed a significant effect of paternal diet-induced obesity on deregulated expression of Peg3, Peg9, Peg10, and the nutrient transporter gene Slc38a2, and aberrant DNA methylation of the Peg9 promoter in F1 placental tissue. Epigenetic changes in Peg9 were also found in sperm from obese fathers. We therefore propose that paternal obesity renders changes in gene expression and/or methylation throughout the placental genome, which could contribute to the reproductive problems related to fertility and to the metabolic, long-term health impact on offspring.

Constitutive promoter methylation of BRCA1 and RAD51C in patients with familial ovarian cancer and early-onset sporadic breast cancer.

Genetic defects in breast cancer (BC) susceptibility genes, most importantly BRCA1 and BRCA2, account for ~40% of hereditary BC and ovarian cancer (OC). Little is known about the contribution of constitutive (soma-wide) epimutations to the remaining cases. We developed bisulfite pyrosequencing assays to screen >600 affected BRCA1/BRCA2 mutation-negative patients from the German Consortium for Hereditary Breast and Ovarian Cancer for constitutive hypermethylation of ATM, BRCA1, BRCA2, RAD51C, PTEN and TP53 in blood cells. In a second step, patients with ≥6% promoter methylation were analyzed by bisulfite plasmid sequencing to demonstrate the presence of hypermethylated alleles (epimutations), indicative of epigenetic gene silencing. Altogether we identified nine (1.4%) patients with constitutive BRCA1 and three (0.5%) with RAD51C hypermethylation. Epimutations were found in both sporadic cases, in particular in 2 (5.5%) of 37 patients with early-onset BC, and familial cases, in particular 4 (10%) of 39 patients with OC. Hypermethylation was always confined to one of the two parental alleles in a subset (12-40%) of the analyzed cells. Because epimutations occurred in cell types from different embryonal layers, they most likely originated in single cells during early somatic development. We propose that analogous to germline genetic mutations constitutive epimutations may serve as the first hit of tumor development. Because the role of constitutive epimutations in cancer development is likely to be largely underestimated, future strategies for effective testing of susceptibility to BC and OC should include an epimutation screen.

Spatial, temporal and interindividual epigenetic variation of functionally important DNA methylation patterns.

DNA methylation is an epigenetic modification that plays an important role in gene regulation. It can be influenced by stochastic events, environmental factors and developmental programs. However, little is known about the natural variation of gene-specific methylation patterns. In this study, we performed quantitative methylation analyses of six differentially methylated imprinted genes (H19, MEG3, LIT1, NESP55, PEG3 and SNRPN), one hypermethylated pluripotency gene (OCT4) and one hypomethylated tumor suppressor gene (APC) in chorionic villus, fetal and adult cortex, and adult blood samples. Both average methylation level and range of methylation variation depended on the gene locus, tissue type and/or developmental stage. We found considerable variability of functionally important methylation patterns among unrelated healthy individuals and a trend toward more similar methylation levels in monozygotic twins than in dizygotic twins. Imprinted genes showed relatively little methylation changes associated with aging in individuals who are >25 years. The relative differences in methylation among neighboring CpGs in the generally hypomethylated APC promoter may not only reflect stochastic fluctuations but also depend on the tissue type. Our results are consistent with the view that most methylation variation may arise after fertilization, leading to epigenetic mosaicism.

Extreme methylation values of imprinted genes in human abortions and stillbirths.

Imprinted genes play an important role in fetal and placental development. Using quantitative bisulfite pyrosequencing assays, we determined the DNA methylation levels at two paternally methylated (H19 and MEG3) and four maternally methylated (LIT1, NESP55, PEG3, and SNRPN) imprinted regions in fetal muscle samples from abortions and stillbirths. Two of 55 (4%) spontaneous abortions and 10 of 57 (18%) stillbirths displayed hypermethylation in multiple genes. Interestingly, none of 34 induced abortions had extreme methylation values in multiple genes. All but two abortions/stillbirths with multiple methylation abnormalities were male, indicating that the male embryo may be more susceptible to excess methylation. Hypermethylation of multiple imprinted genes is consistent with stochastic failures of the mechanism, which normally protects the hypomethylated allele from de novo methylation after fertilization. Two of six informative abortions/stillbirths with H19 hypermethylation revealed significant biallelic expression of the autocrine growth factor IGF2. In two other cases hypermethylation of MEG3 was associated with transcriptional down-regulation. We propose that primary epimutations resulting in inappropriate methylation and expression patterns of imprinted genes may contribute to both normal human variation and disease, in particular spontaneous pregnancy loss.

Quantitative methylation analysis of developmentally important genes in human pregnancy losses after ART and spontaneous conception.

To study possible effects of assisted reproductive technologies (ART) on epigenetic reprogramming, we have analyzed the DNA methylation levels of differentially methylated regions (DMRs) of seven imprinted genes (H19, MEG3, LIT1, MEST, NESP55, PEG3 and SNRPN) as well as the promoter regions of the pluripotency gene NANOG and the tumor suppressor gene APC in chorionic villus samples (CVS) of 42 spontaneous miscarriages and stillbirths after ART and 29 abortions/stillbirths after spontaneous conception. We did not find an increased rate of faulty methylation patterns after ART, but significant and trend differences (ROC curve analysis, Wilcoxon test) in the methylation levels of LIT1 (P = 0.006) and H19 (P = 0.085) between ART and non-ART samples. With the possible exception of NANOG, we did not observe a gestational age effect on the methylation levels of the studied genes. The frequency of extreme methylation values in PEG3 and APC was markedly higher than in the other studied genes, indicating an increased susceptibility of some genes to epigenetic alterations. Most methylation abnormalities in CVS represented either hypermethylated DMRs of paternally and maternally imprinted genes or hypomethylated promoters of non-imprinted genes. The observed methylation abnormalities (mosaicism) are consistent with methylation reprogramming defects during early embryogenesis.

In vitro maturation of oocytes is not associated with altered deoxyribonucleic acid methylation patterns in children from in vitro fertilization or intracytoplasmic sperm injection.

OBJECTIVE: To study the possible transmission, to the next generation, of epigenetic defects associated with in vitro maturation (IVM) of human oocytes. DESIGN: Case-control study using epigenetic data. SETTING: Two collaborating university departments. PATIENT(S): Eleven IVM newborns and 19 controls, conceived by conventional assisted reproduction. INTERVENTION(S): Chorionic villus and cord-blood sampling. MAIN OUTCOME MEASURE(S): Using bisulfite pyrosequencing, we have measured average methylation levels of 6 imprinted (LIT1, MEG, MEST, NESPas, PEG3, and SNRPN), 5 tumor-suppressor (APC, ATM, BRCA1, RAD51C, and TP53), 2 pluripotency (NANOG and OCT4), and 2 metabolic (LEP and NR3C1) genes, as well as 2 repetitive elements (ALU and LINE1) in 2 tissues of IVM and control neonates. Using deep bisulfite sequencing, we have determined methylation patterns of many individual DNA molecules to detect rare RAD51C epimutations (allele methylation errors). RESULT(S): No statistically significant impact was found of IVM on chorionic villus and cord-blood DNA methylation at the studied developmentally important genes and interspersed repeats. The RAD51C epimutation rate was low (0.5% ± 0.1%) in all analyzed samples. CONCLUSION(S): IVM-induced epigenetic changes in offspring, if any, are relatively small in magnitude and/or infrequent.

Metabolic programming of MEST DNA methylation by intrauterine exposure to gestational diabetes mellitus.

Epigenetic processes are primary candidates when searching for mechanisms that can stably modulate gene expression and metabolic pathways according to early life conditions. To test the effects of gestational diabetes mellitus (GDM) on the epigenome of the next generation, cord blood and placenta tissue were obtained from 88 newborns of mothers with dietetically treated GDM, 98 with insulin-dependent GDM, and 65 without GDM. Bisulfite pyrosequencing was used to compare the methylation levels of seven imprinted genes involved in prenatal and postnatal growth, four genes involved in energy metabolism, one anti-inflammatory gene, one tumor suppressor gene, one pluripotency gene, and two repetitive DNA families. The maternally imprinted MEST gene, the nonimprinted glucocorticoid receptor NR3C1 gene, and interspersed ALU repeats showed significantly decreased methylation levels (4-7 percentage points for MEST, 1-2 for NR3C1, and one for ALUs) in both GDM groups, compared with controls, in both analyzed tissues. Significantly decreased blood MEST methylation (3 percentage points) also was observed in adults with morbid obesity compared with normal-weight controls. Our results support the idea that intrauterine exposure to GDM has long-lasting effects on the epigenome of the offspring. Specifically, epigenetic malprogramming of MEST may contribute to obesity predisposition throughout life.

Sequence-based bioinformatic prediction and QUASEP identify genomic imprinting of the KCNK9 potassium channel gene in mouse and human.

Genomic imprinting is the epigenetic marking of gene subsets resulting in monoallelic or predominant expression of one of the two parental alleles according to their parental origin. We describe the systematic experimental verification of a prioritized 16 candidate imprinted gene set predicted by sequence-based bioinformatic analyses. We used Quantification of Allele-Specific Expression by Pyrosequencing (QUASEP) and discovered maternal-specific imprinted expression of the Kcnk9 gene as well as strain-dependent preferential expression of the Rarres1 gene in E11.5 (C57BL/6 x Cast/Ei)F1 and informative (C57BL/6 x Cast/Ei) x C57BL/6 backcross mouse embryos. For the remaining 14 candidate imprinted genes, we observed biallelic expression. In adult mouse tissues, we found that Kcnk9 expression was restricted to the brain and also was maternal-specific. QUASEP analysis of informative human fetal brain samples further demonstrated maternal-specific imprinted expression of the human KCNK9 orthologue. The CpG islands associated with the mouse and human Kcnk9/KCNK9 genes were not differentially methylated, but strongly hypomethylated. Thus, we speculate that mouse Kcnk9 imprinting may be regulated by the maternal germline differentially methylated region in Peg13, an imprinted non-coding RNA gene in close proximity to Kcnk9 on distal mouse chromosome 15. Our data have major implications for the proposed role of Kcnk9 in neurodevelopment, apoptosis and tumourigenesis, as well as for the efficiency of sequence-based bioinformatic predictions of novel imprinted genes.